- standard electroporation protocol
- used electro competend DH5alpha from NEB
- streaked out on LB-plates with Cam
(created page) |
|||
(6 intermediate revisions by 2 users not shown) | |||
Line 4: | Line 4: | ||
<body> | <body> | ||
<div class="container"> | <div class="container"> | ||
+ | <div id="title" style="background-image: url(https://static.igem.org/mediawiki/2017/9/9a/T--Bielefeld-CeBiTec--title-img-labjournal.jpg);"> | ||
+ | <img src="https://static.igem.org/mediawiki/2017/9/9a/T--Bielefeld-CeBiTec--title-img-labjournal.jpg"> | ||
+ | <div id="title-bg"> | ||
+ | <div id="title-text"> | ||
+ | Labjournal | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | |||
+ | |||
+ | <!--generated by the Labnotesgenerator from Bielefeld-CeBiTec 2017--> | ||
+ | <meta charset="UTF-8"><link rel="stylesheet" type="text/css" href="labnotes_css.css"> | ||
+ | <script src="https://ajax.googleapis.com/ajax/libs/jquery/3.2.1/jquery.min.js"></script> | ||
+ | <script src="labnotes_js.js"></script> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-date"> | ||
+ | <h3> 2017-04-03 - 2017-04-09 </h3> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> getting positive clones for further work </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Yannic Kerkhoff</p> | ||
+ | <p><b>Superior experiment:</b> electroporation transformation of BBa_K1416000</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> standard electroporation protocol</li> | ||
+ | <ul> | ||
+ | <li> used electro competend DH5alpha from NEB</li> | ||
+ | <li> streaked out on LB-plates with Cam</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> getting positive clones for further work </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Yannic Kerkhoff</p> | ||
+ | <p><b>Superior experiment:</b> electroporation transformation of BBa_KJ36848</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> standard electroporation protocol</li> | ||
+ | <ul> | ||
+ | <li> used electro competend DH5alpha from NEB</li> | ||
+ | <li> streaked out on LB-plates with Cam</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> multiplication of plasmids containing the Biobrick </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Yannic Kerkhoff</p> | ||
+ | <p><b>Superior experiment:</b> preculture of transformed BBa_K1416000</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> standard procedure</li> | ||
+ | <ul> | ||
+ | <li> 1,25µl Cam in 5mL LB</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-date"> | ||
+ | <h3> 2017-04-10 - 2017-04-16 </h3> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> getting plasmids containing the Biobrick for further experiments </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Yannic Kerkhoff</p> | ||
+ | <p><b>Superior experiment:</b> plasmid isolation of preculture of BBa_K1416000</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> standard protocol</li> | ||
+ | <ul> | ||
+ | <li> 517,9 ng/µL</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> multiplication of plasmids containing the Biobrick </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Yannic Kerkhoff</p> | ||
+ | <p><b>Superior experiment:</b> preculture of transformed BBa_J36848</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> standard procedure</li> | ||
+ | <ul> | ||
+ | <li> 1,25µl Cam in 5mL LB</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> getting plasmids containing the Biobrick for further experiments </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Yannic Kerkhoff</p> | ||
+ | <p><b>Superior experiment:</b> plasmid isolation of preculture of BBa_J36848</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> standard protocol</li> | ||
+ | <ul> | ||
+ | <li> 231,8 ng/µL</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-date"> | ||
+ | <h3> 2017-05-29 - 2017-06-04 </h3> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> getting positive clones for further work </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Yannic Kerkhoff</p> | ||
+ | <p><b>Superior experiment:</b> heatshock transformation of BBa_E0400</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> standard heatshock protocol</li> | ||
+ | <ul> | ||
+ | <li> used own produced chemo competend DH5alpha</li> | ||
+ | <li> streaked out on LB-plates with Amp</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-date"> | ||
+ | <h3> 2017-06-05 - 2017-06-11 </h3> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> multiplication of plasmids containing the Biobrick </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Yannic Kerkhoff</p> | ||
+ | <p><b>Superior experiment:</b> preculture of transformed BBa_E0400</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> standard procedure</li> | ||
+ | <ul> | ||
+ | <li> 5,0µl Amp in 5mL LB</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> getting plasmids containing the Biobrick for further experiments </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Yannic Kerkhoff</p> | ||
+ | <p><b>Superior experiment:</b> plasmid isolation of preculture of BBa_E0400</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> standard protocol</li> | ||
+ | <ul> | ||
+ | <li> 88,4 ng/µL</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> getting positive clones for further work </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Yannic Kerkhoff</p> | ||
+ | <p><b>Superior experiment:</b> heatshock transformation of BBa_K525998</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> standard heatshock protocol</li> | ||
+ | <ul> | ||
+ | <li> used own produced chemo competend DH5alpha</li> | ||
+ | <li> streaked out on LB-plates with Cam</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> multiplication of plasmids containing the Biobrick </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Yannic Kerkhoff</p> | ||
+ | <p><b>Superior experiment:</b> preculture of transformed BBa_K525998</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> standard procedure</li> | ||
+ | <ul> | ||
+ | <li> 1,25µl Cam in 5mL LB</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> getting plasmids containing the Biobrick for further experiments </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Yannic Kerkhoff</p> | ||
+ | <p><b>Superior experiment:</b> plasmid isolation of preculture of BBa_K525998</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> standard protocol</li> | ||
+ | <ul> | ||
+ | <li> 140,9 ng/µL</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-date"> | ||
+ | <h3> 2017-06-12 - 2017-06-18 </h3> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Assemble the fragments to get the Part BBa_K2201220 </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Yannic Kerkhoff</p> | ||
+ | <p><b>Superior experiment:</b> Gibson assembly of F1,F3,F5</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> standard <a target="blank" href="https://static.igem.org/mediawiki/2017/5/55/T--Bielefeld-CeBiTec--protocol_gibson_assembly.pdf">Gibson Assembly</a> protocol</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Cellgrowth with the barnaseplasmid </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Olga Schmidt, Denise Kerkhoff, Christina Drake</p> | ||
+ | <p><b>Superior experiment:</b> Overnightculture of barnase</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> 3 ml <a target="blank" href="https://static.igem.org/mediawiki/2017/a/a7/T--Bielefeld-CeBiTec--protocol_LB.pdf">LB-medium</a> and 0,75 µl chloramphenicol</li> | ||
+ | <li> inkubation overnight 37°C, 140rpm</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> To get and purified the plasmid </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Christina Drake</p> | ||
+ | <p><b>Superior experiment:</b> Plasmidisolation of barnase-plasmid</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> protocol from Macherley-Nagel: Plasmid DNA purification, protocol 5: NucleoSpin</li> | ||
+ | <li> concentration measurement by nanoprop</li> | ||
+ | <li> sequenzingorder</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Duplicate the parts </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Christina Drake, Denise Kerkhoff</p> | ||
+ | <p><b>Superior experiment:</b> Transformation via Distribution of the parts BBA_J04450, BBa_I746909, BBa_K80800 and BBa_psB1K5</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> <a target="blank" href="https://static.igem.org/mediawiki/2017/a/ab/T--Bielefeld-CeBiTec--protocol_electrotrafo.pdf">transformation via electroporation</a></li> | ||
+ | <li> plated out: BBa_J04450 on tetracycline, BBa_I74909 and BBa_K808000 on chloramphenicol, BBa_psB1K5 on kanamycin</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Duplicate the part </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Christina Drake, Denise Kerkhoff</p> | ||
+ | <p><b>Superior experiment:</b> Transformation of BBa_psB6A1</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> <a target="blank" href="https://static.igem.org/mediawiki/2017/a/ab/T--Bielefeld-CeBiTec--protocol_electrotrafo.pdf">transformation via electroporation</a></li> | ||
+ | <li> plated out on amphenicol</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Cellgrowth with the plasmids </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Denise Kerkhoff, Christina Drake</p> | ||
+ | <p><b>Superior experiment:</b> Overnightculture of BBA_J04450, BBa_I746909 and BBa_K808000</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> 3 ml <a target="blank" href="https://static.igem.org/mediawiki/2017/a/a7/T--Bielefeld-CeBiTec--protocol_LB.pdf">LB-medium</a> and at BBA_J04450 3µl tetracycline and atBBa_I746909 and BBa_K808000 0,75 µl chloramphenicol</li> | ||
+ | <li> inkubation overnight 37°C, 140rpm</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> To get and purified the plasmid </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Christina Drake</p> | ||
+ | <p><b>Superior experiment:</b> Plasmidisolation of BBA_J04450, BBa_I746909 and BBa_K80800-plasmids</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> protocol from Macherley-Nagel: Plasmid DNA purification, protocol 5: NucleoSpin</li> | ||
+ | <li> concentration measurement by nanoprop:</li> | ||
+ | <ul> | ||
+ | <li> BBA_J04450 26ng/µl and 25,7ng/µl</li> | ||
+ | <li> BBa_I746909 90,2ng/µl</li> | ||
+ | <li> 104,3 ng/µl -BBa_K808000 118,3 ng/µl</li> | ||
+ | </ul> | ||
+ | <li> and 146,6 ng/µl</li> | ||
+ | <li> sequenzingorder</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-date"> | ||
+ | <h3> 2017-06-19 - 2017-06-25 </h3> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Cellgrowth with the plasmid </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Denise Kerkhoff</p> | ||
+ | <p><b>Superior experiment:</b> Overnightculture of BBA_psB3K5</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> 3 ml <a target="blank" href="https://static.igem.org/mediawiki/2017/a/a7/T--Bielefeld-CeBiTec--protocol_LB.pdf">LB-medium</a> and 2,5 µl kanamycin</li> | ||
+ | <li> inkubation overnight 37°C, 140rpm</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> getting positive clones for further work </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Yannic Kerkhoff</p> | ||
+ | <p><b>Superior experiment:</b> heatshock transformation of assembled fragments F5,F2,F4</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> standard heatshock protocol</li> | ||
+ | <ul> | ||
+ | <li> used own produced chemo competend DH5alpha</li> | ||
+ | <li> streaked out on LB-plates with Cam</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> multiplication, verification and purification of the needed fragment F2: GFPLinker2 </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Yannic Kerkhoff</p> | ||
+ | <p><b>Superior experiment:</b> Q5-PCR, gel and PCR-cleanUp of F2</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> standard Q5-PCR protocol</li> | ||
+ | <ul> | ||
+ | <li> template: BBa_E0400</li> | ||
+ | <li> primer fwd: 17gk</li> | ||
+ | <li> primer rev: 17gn</li> | ||
+ | <li> annealing temperature: 58°C</li> | ||
+ | <li> extension time: 50 seconds</li> | ||
+ | </ul> | ||
+ | <li> standard PCR-cleanUp protocol</li> | ||
+ | <ul> | ||
+ | <li> 125,8 ng/µL</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> multiplication, verification and purification of the needed fragment F4: StrepLinker2 </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Yannic Kerkhoff</p> | ||
+ | <p><b>Superior experiment:</b> Q5-PCR, gel and PCR-cleanUp of F4</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> standard Q5-PCR protocol</li> | ||
+ | <ul> | ||
+ | <li> template: BBa_KJ36848</li> | ||
+ | <li> primer fwd: 17gp</li> | ||
+ | <li> primer rev: 17gq</li> | ||
+ | <li> annealing temperature: 61°C</li> | ||
+ | <li> extension time: 30 seconds</li> | ||
+ | </ul> | ||
+ | <li> standard PCR-cleanUp protocol</li> | ||
+ | <ul> | ||
+ | <li> 127,0 ng/µL</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> multiplication, verification and purification of the needed fragment F5: linearized pSB1C3 </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Yannic Kerkhoff</p> | ||
+ | <p><b>Superior experiment:</b> Q5-PCR, gel and PCR-cleanUp of F5</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> standard Q5-PCR protocol</li> | ||
+ | <ul> | ||
+ | <li> template: K1416000</li> | ||
+ | <li> primer fwd: 17gj</li> | ||
+ | <li> primer rev: 17gl</li> | ||
+ | <li> annealing temperature: 59°C</li> | ||
+ | <li> extension time: 120 seconds</li> | ||
+ | </ul> | ||
+ | <li> standard PCR-cleanUp protocol</li> | ||
+ | <ul> | ||
+ | <li> 258,4 ng/µL</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Assemble the fragments to get the Part BBa_K2201221 </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Yannic Kerkhoff</p> | ||
+ | <p><b>Superior experiment:</b> Gibson assembly of F2,F4,F5</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> standard <a target="blank" href="https://static.igem.org/mediawiki/2017/5/55/T--Bielefeld-CeBiTec--protocol_gibson_assembly.pdf">Gibson Assembly</a> protocol</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Screening for positive transformations containing the Biobrick </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Yannic Kerkhoff</p> | ||
+ | <p><b>Superior experiment:</b> GoTaq PCR of 7 clones of BBa_K2201221</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> standard GoTaq-protocol</li> | ||
+ | <ul> | ||
+ | <li> template: colonie pcr clones of GA_K2201221</li> | ||
+ | <li> primer fwd: Präfix_fwd</li> | ||
+ | <li> primer rev: Suffix_rev</li> | ||
+ | <li> annealing temperature: 56°C</li> | ||
+ | <li> extension time: 40 seconds</li> | ||
+ | </ul> | ||
+ | <li> positive clone 2</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> multiplication of plasmids containing the Biobrick </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Yannic Kerkhoff</p> | ||
+ | <p><b>Superior experiment:</b> preculture of the positive clone 2 of BBa_K2201221</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> standard procedure</li> | ||
+ | <ul> | ||
+ | <li> 1,25µl Cam in 5mL LB</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> getting plasmids containing the Biobrick for further experiments </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Yannic Kerkhoff</p> | ||
+ | <p><b>Superior experiment:</b> plasmid isolation of preculture of BBa_K2201200 clone 20</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> standard protocol</li> | ||
+ | <ul> | ||
+ | <li> 240,6 ng/µL</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> getting plasmids containing the Biobrick for further experiments </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Yannic Kerkhoff</p> | ||
+ | <p><b>Superior experiment:</b> plasmid isolation of preculture of BBa_K2201221 clone 2</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> standard protocol</li> | ||
+ | <ul> | ||
+ | <li> 208,8 ng/µL</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-date"> | ||
+ | <h3> 2017-06-26 - 2017-07-02 </h3> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Check the sequence </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Christina Drake</p> | ||
+ | <p><b>Superior experiment:</b> Sequenzingorder barnase</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> concentration measurement by nanoprop: 177 ng/µl</li> | ||
+ | <li> result: sequence was not barnase</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Plasmidisolation and gibson assembly of pSB1C3-PlacUV5_PtNTT2 c30 </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Camilla Maerz</p> | ||
+ | <p><b>Superior experiment:</b> Construction of pSB1C3-PlacUV5_PtNTT2</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> Plasmid DNA purification kit</li> | ||
+ | <ul> | ||
+ | <li> Nucleo spin plasmid protocol</li> | ||
+ | </ul> | ||
+ | <li> <a target="blank" href="https://static.igem.org/mediawiki/2017/5/55/T--Bielefeld-CeBiTec--protocol_gibson_assembly.pdf">Gibson Assembly</a> protocol</li> | ||
+ | <li> <a target="blank" href="https://static.igem.org/mediawiki/2017/a/a0/T--Bielefeld-CeBiTec--protocol_heatshock.pdf">transformation via heat shock</a> protocol in <i>E. coli</i> DH5<i>alpha</i></li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Colony PCR of T7RNAP from <i>E. coli</i> KRX </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Olga Schmidt</p> | ||
+ | <p><b>Superior experiment:</b> Construction of the positive selection plasmid</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> PCR with Grimson Taq polymerase (per reaction, 25 µL):</li> | ||
+ | <ul> | ||
+ | <li> 5 µL 5x Buffer</li> | ||
+ | <li> 2.5 µL dNTPs (2 mM)</li> | ||
+ | <li> 0.5 µL 3'-Primer (10 mM)</li> | ||
+ | <li> 0.5 µL 5'-Primer (10 mM)</li> | ||
+ | <li> 0.125 µL Grimson Taq polymerase</li> | ||
+ | <li> 1 picked bacterial colony as template</li> | ||
+ | <li> ad 25 µL H20</li> | ||
+ | </ul> | ||
+ | <li> Primer annealing: 64 °C</li> | ||
+ | <li> Elongation: 150 s</li> | ||
+ | <li> Agarose gel electrophoresis (1%)</li> | ||
+ | <ul> | ||
+ | <li> 100 V, 30 min</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Isolate T7RNAP from the chromosome of KRX-e.coli </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Christina Drake</p> | ||
+ | <p><b>Superior experiment:</b> PCR of T7RNAP from KRX-e.coli</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> <a target="blank" href="https://static.igem.org/mediawiki/2017/8/88/T--Bielefeld-CeBiTec--protocol_PCR_Q5_HF_Polymerase.pdf">Q5 High-Fidelity PCR</a></li> | ||
+ | <ul> | ||
+ | <li> annealingtemparatur 64°C, elogationtime 1 min</li> | ||
+ | </ul> | ||
+ | <li> 1%-agarose-<a target="blank" href="https://static.igem.org/mediawiki/2017/d/d4/T--Bielefeld-CeBiTec--protocol_TAE_buffer.pdf">TAE</a>-gel</li> | ||
+ | <ul> | ||
+ | <li> 1kb Marker Geneuler and 5 µl sample with 1 µl 6*Loading Dye</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Plated out Barnase from strain collection </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Christina Drake</p> | ||
+ | <p><b>Superior experiment:</b> Plated out Barnase from strain collection</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> solved DNA in 100 µl <a target="blank" href="https://static.igem.org/mediawiki/2017/f/fe/T--Bielefeld-CeBiTec--protocol_SOC.pdf">SOC-medium</a></li> | ||
+ | <li> plated out on chloramphenicolplate</li> | ||
+ | <li> overnightincubation at 37°C</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Colony PCR of pK18mobsacB-del_codA </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Camilla Maerz</p> | ||
+ | <p><b>Superior experiment:</b> Construction of pK18mobsacB_codA_del</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> Go Tag (Promega) protocol</li> | ||
+ | <ul> | ||
+ | <li> Primer: VR, VF2</li> | ||
+ | <li> Primer annealing: 56 °C</li> | ||
+ | <li> Elongation: 140 s</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> PCR of pSB1C3 </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Olga Schmidt</p> | ||
+ | <p><b>Superior experiment:</b> General</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> PCR</li> | ||
+ | <ul> | ||
+ | <li> Q5 High-fidelily (NEB) protocol</li> | ||
+ | <li> Denaturation: 64 °C</li> | ||
+ | <li> Elongation: 60 s</li> | ||
+ | </ul> | ||
+ | <li> Agarose gel electrophoresis (1%)</li> | ||
+ | <ul> | ||
+ | <li> 100 V, 30 min</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Gibson assembly of T7RNAP in pSB1C3 </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Olga Schmidt</p> | ||
+ | <p><b>Superior experiment:</b> Construction of the selection plasmid</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> <a target="blank" href="https://static.igem.org/mediawiki/2017/5/55/T--Bielefeld-CeBiTec--protocol_gibson_assembly.pdf">Gibson Assembly</a> protocol</li> | ||
+ | <li> <a target="blank" href="https://static.igem.org/mediawiki/2017/a/a0/T--Bielefeld-CeBiTec--protocol_heatshock.pdf">transformation via heat shock</a> protocol in <i>E. coli</i> BL21</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Colony PCR of pSB1C3_T7RNAP c1-8 </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Olga Schmidt</p> | ||
+ | <p><b>Superior experiment:</b> Construction of the selection plasmid</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> PCR</li> | ||
+ | <ul> | ||
+ | <li> Q5 High-fidelily (NEB) protocol</li> | ||
+ | <li> Denaturation: 64 °C</li> | ||
+ | <li> Elongation: 80 s</li> | ||
+ | </ul> | ||
+ | <li> Agarose gel electrophoresis (1%)</li> | ||
+ | <ul> | ||
+ | <li> 100 V, 30 min</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Colony PCR of barnase (26.06.2017) c1&2 </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Olga Schmidt</p> | ||
+ | <p><b>Superior experiment:</b> Construction of selection plasmid</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> PCR</li> | ||
+ | <ul> | ||
+ | <li> Go Tag (Promega) protocol</li> | ||
+ | <li> Danaturation: 58 °C</li> | ||
+ | <li> Elongation: 35 s</li> | ||
+ | </ul> | ||
+ | <li> Agarose gel electrophoresis (1%)</li> | ||
+ | <ul> | ||
+ | <li> 100 V, 30 min</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-date"> | ||
+ | <h3> 2017-07-03 - 2017-07-09 </h3> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Check the correct barnasegen </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Christina Drake</p> | ||
+ | <p><b>Superior experiment:</b> PCR of barnase</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> Go-Taq-protocol</li> | ||
+ | <ul> | ||
+ | <li> annealingtemparatur 58°C, elogationtime 30 s</li> | ||
+ | </ul> | ||
+ | <li> 1%-agarose-<a target="blank" href="https://static.igem.org/mediawiki/2017/d/d4/T--Bielefeld-CeBiTec--protocol_TAE_buffer.pdf">TAE</a>-gel</li> | ||
+ | <ul> | ||
+ | <li> 100bp Marker Geneuler and 5 µl sample with 1 µl 6*Loading Dye</li> | ||
+ | <li> result: PCR-product could not be barnase</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-date"> | ||
+ | <h3> 2017-07-10 - 2017-07-16 </h3> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Biobrick Assembly of 2B1 </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Maximilian Edich</p> | ||
+ | <p><b>Superior experiment:</b> Biobrick Construction with RuBisCo Parts</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> <a target="blank" href="https://static.igem.org/mediawiki/2017/8/84/T--Bielefeld-CeBiTec--protocol_Standard_Biobrick.pdf">BioBrick Assembly</a></li> | ||
+ | <ul> | ||
+ | <li> RuBisCoTAG mRFP as upstream part, T7-Terminator as downstream part, pSB1A3 as destination plasmid</li> | ||
+ | </ul> | ||
+ | <li> <a target="blank" href="https://static.igem.org/mediawiki/2017/a/a0/T--Bielefeld-CeBiTec--protocol_heatshock.pdf">transformation via heat shock</a> and DH5α Cells</li> | ||
+ | <ul> | ||
+ | <li> used 5µl from the ligation</li> | ||
+ | </ul> | ||
+ | <li> plated out on Amp plates and let them grow over night</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Check the sequence </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Christina Drake</p> | ||
+ | <p><b>Superior experiment:</b> Sequenzingorder barnase</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> result: sequence was not barnase</li> | ||
+ | </ul> | ||
+ | <li> >16.07.2017</li> | ||
+ | <li> tRNA</li> | ||
+ | <li> Overnightculture of TyrRS-Heidelberg-plasmid</li> | ||
+ | <li> CDR</li> | ||
+ | <li> Cellgrowth with the TyrRS-Heidelberg-plasmid</li> | ||
+ | <ul> | ||
+ | <li> 3 ml <a target="blank" href="https://static.igem.org/mediawiki/2017/a/a7/T--Bielefeld-CeBiTec--protocol_LB.pdf">LB-medium</a> and 0,75 µl chloramphenicol</li> | ||
+ | <li> inkubation overnight 37°C, 140rpm</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Transformation of T7-Promotor </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Maximilian Edich</p> | ||
+ | <p><b>Superior experiment:</b> Biobrick Construction with RuBisCo Parts</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> <a target="blank" href="https://static.igem.org/mediawiki/2017/a/ab/T--Bielefeld-CeBiTec--protocol_electrotrafo.pdf">transformation via electroporation</a> of cells with T7-Promotor</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Plasmidisolation of TyrRS-Heidelberg plasmid </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Olga Schmidt</p> | ||
+ | <p><b>Superior experiment:</b> Construction of selection plasmid</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> Plasmid DNA purification kit (Macherey-Nagel)</li> | ||
+ | <ul> | ||
+ | <li> Nucleo spin plasmid protocol</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Preculture of colonies with T7-Promotor </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Maximilian Edich</p> | ||
+ | <p><b>Superior experiment:</b> Biobrick Construction with RuBisCo Parts</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> used 5µl <a target="blank" href="https://static.igem.org/mediawiki/2017/a/a7/T--Bielefeld-CeBiTec--protocol_LB.pdf">LB media</a> and kanamycin</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-date"> | ||
+ | <h3> 2017-07-17 - 2017-07-23 </h3> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Isolate the tRNA </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Christina Drake</p> | ||
+ | <p><b>Superior experiment:</b> PCR of TyrRS-Heidelberg-plasmid</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> plasmidisolation with MN protocol 5</li> | ||
+ | <li> <a target="blank" href="https://static.igem.org/mediawiki/2017/8/88/T--Bielefeld-CeBiTec--protocol_PCR_Q5_HF_Polymerase.pdf">Q5 High-Fidelity PCR</a></li> | ||
+ | <ul> | ||
+ | <li> annealingtemparatur 53°C, elogationtime 20s</li> | ||
+ | </ul> | ||
+ | <li> Gibbsonassembly with 5µl backbone and 0.5 µl insert</li> | ||
+ | <li> <a target="blank" href="https://static.igem.org/mediawiki/2017/a/a0/T--Bielefeld-CeBiTec--protocol_heatshock.pdf">transformation via heat shock</a></li> | ||
+ | <li> overnight incubation at 37°C</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Isolation of T7-Promotor </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Laura Schlueter</p> | ||
+ | <p><b>Superior experiment:</b> Biobrick Construction with RuBisCo Parts</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> Nucleospin Plasmidisolation protocol</li> | ||
+ | <ul> | ||
+ | <li> got four products with the concentrations 50.1 ng/µl, 16.3 ng/µl, 24,2 ng/µl and 2.1 ng/µl</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Colony PCR with 2B1 Colonies </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Maximilian Edich</p> | ||
+ | <p><b>Superior experiment:</b> Biobrick Construction with RuBisCo Parts</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> <a target="blank" href="https://static.igem.org/mediawiki/2017/c/cc/T--Bielefeld-CeBiTec--protocol_colonyPCR_GoTaq_G2.pdf">GoTaq® G2 PCR</a></li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Biobrickassembly of 2B2 to 2B8, B1 and A1 </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Maximilian Edich</p> | ||
+ | <p><b>Superior experiment:</b> Biobrick Construction with RuBisCo Parts</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> <a target="blank" href="https://static.igem.org/mediawiki/2017/8/84/T--Bielefeld-CeBiTec--protocol_Standard_Biobrick.pdf">BioBrick Assembly</a></li> | ||
+ | <ul> | ||
+ | <li> digest and ligate RuBisCo parts as upstreampart and T7-Terminator/Promotor as downstreampart and pSB1A3 as destination plasmid</li> | ||
+ | <ul> | ||
+ | <li> ligate TAG2 and T7-Terminator to 2B2</li> | ||
+ | <li> ligate TAG111 and T7-Terminator to 2B3</li> | ||
+ | <li> ligate TAG474 and T7-Terminator to 2B4</li> | ||
+ | <li> ligate TAG2+TAG111 and T7-Terminator to 2B5</li> | ||
+ | <li> ligate TAG2+TAG474 and T7-Terminator to 2B6</li> | ||
+ | <li> ligate TAG111+TAG474 and T7-Terminator to 2B7</li> | ||
+ | <li> ligate TAG2+TAG111+TAG474 and T7-Terminator to 2B8</li> | ||
+ | <li> ligate RuBisCo mRFP and T7-Terminator to B1</li> | ||
+ | <li> ligate Carboxysome and T7-Promotor to A1</li> | ||
+ | </ul> | ||
+ | <li> preculture of positive 2B1 colony</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Transformation of test devices </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Camilla Maerz</p> | ||
+ | <p><b>Superior experiment:</b> Interlab study</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> dilute test devives (distribution plate 6 & 7) in 10 µL ddH20</li> | ||
+ | <li> Transformation</li> | ||
+ | <ul> | ||
+ | <li> 1 µL plasmid from each plate</li> | ||
+ | <ul> | ||
+ | <li> <a target="blank" href="https://static.igem.org/mediawiki/2017/a/a0/T--Bielefeld-CeBiTec--protocol_heatshock.pdf">transformation via heat shock</a> protocol in <i>E. coli</i> XL1-blue</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Transformation of 2B2-2B8, B1 and A1 </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Maximilian Edich</p> | ||
+ | <p><b>Superior experiment:</b> Biobrick Construction with RuBisCo Parts</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> <a target="blank" href="https://static.igem.org/mediawiki/2017/a/a0/T--Bielefeld-CeBiTec--protocol_heatshock.pdf">transformation via heat shock</a></li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Plasmidisolation of 2B1 </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Maximilian Edich</p> | ||
+ | <p><b>Superior experiment:</b> Biobrick Construction with RuBisCo Parts</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> Nucleospin Plasmidisolation protocol</li> | ||
+ | <ul> | ||
+ | <li> got 47 ng/µl and 41 ng/µl</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Reverse the insert </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Christina Drake</p> | ||
+ | <p><b>Superior experiment:</b> PCR of T7GFP</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> <a target="blank" href="https://static.igem.org/mediawiki/2017/8/88/T--Bielefeld-CeBiTec--protocol_PCR_Q5_HF_Polymerase.pdf">Q5 High-Fidelity PCR</a></li> | ||
+ | <ul> | ||
+ | <li> Insert: annealingtemparatur 60°C,</li> | ||
+ | <li> Backbone:elogationtime 30s: annealingtemparatur 60°C, elogationtime 1 min</li> | ||
+ | <li> 1%-agarose-<a target="blank" href="https://static.igem.org/mediawiki/2017/d/d4/T--Bielefeld-CeBiTec--protocol_TAE_buffer.pdf">TAE</a>-gel</li> | ||
+ | </ul> | ||
+ | <li> result: PCR didn`t work</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Reverse the insert </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Christina Drake</p> | ||
+ | <p><b>Superior experiment:</b> PCR of T7GFP</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> 1%-agarose-<a target="blank" href="https://static.igem.org/mediawiki/2017/d/d4/T--Bielefeld-CeBiTec--protocol_TAE_buffer.pdf">TAE</a>-gel</li> | ||
+ | <li> result: PCR did work</li> | ||
+ | <li> <a target="blank" href="https://static.igem.org/mediawiki/2017/5/55/T--Bielefeld-CeBiTec--protocol_gibson_assembly.pdf">Gibson Assembly</a></li> | ||
+ | <li> <a target="blank" href="https://static.igem.org/mediawiki/2017/a/a0/T--Bielefeld-CeBiTec--protocol_heatshock.pdf">transformation via heat shock</a></li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> PCR of BBa_I746909 and pSB1C3 </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Olga Schmidt</p> | ||
+ | <p><b>Superior experiment:</b> Integration of CDS of BBa_I746909 in pSB1C3</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> PCR of I746909</li> | ||
+ | <ul> | ||
+ | <li> Q5 High-fidelily (NEB) protocol</li> | ||
+ | <li> Primer: 17lk & 17lj</li> | ||
+ | <li> Denaturation: 60 °C</li> | ||
+ | <li> Elongation: 40 s</li> | ||
+ | </ul> | ||
+ | <li> PCR of pSB1C3</li> | ||
+ | <ul> | ||
+ | <li> Q5 High-fidelily (NEB) protocol</li> | ||
+ | <li> Primer: 17ll & 17li</li> | ||
+ | <li> Denaturation: 60 °C</li> | ||
+ | <li> Elongation: 70 s</li> | ||
+ | </ul> | ||
+ | <li> Agarose gel electrophoresis (1%)</li> | ||
+ | <ul> | ||
+ | <li> 100 V, 30 min</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Preparation of over-night culture </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Camilla Maerz</p> | ||
+ | <p><b>Superior experiment:</b> Interlab study</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> Over-night culture of negative control, positive control, test device 1-6</li> | ||
+ | <ul> | ||
+ | <li> <a target="blank" href="https://static.igem.org/mediawiki/2017/a/a7/T--Bielefeld-CeBiTec--protocol_LB.pdf">LB-media</a> + Cm were inoculated</li> | ||
+ | <li> Cultures were incubated over night (37°C, 200 rpm)</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-date"> | ||
+ | <h3> 2017-07-24 - 2017-07-30 </h3> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Plasmidisolation of pSB1C3_I746909 </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Olga Schmidt</p> | ||
+ | <p><b>Superior experiment:</b> Integration of CDS of BBa_I746909 in pSB1C3</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> Plasmid DNA purification kit (Macherey-Nagel)</li> | ||
+ | <ul> | ||
+ | <li> Nucleo spin plasmid protocol</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Repeat the degestion and transformation from the 18th and 19th july </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Maximilian Edich</p> | ||
+ | <p><b>Superior experiment:</b> Biobrick Construction with RuBisCo Parts</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> <a target="blank" href="https://static.igem.org/mediawiki/2017/8/84/T--Bielefeld-CeBiTec--protocol_Standard_Biobrick.pdf">BioBrick Assembly</a></li> | ||
+ | <li> <a target="blank" href="https://static.igem.org/mediawiki/2017/a/a0/T--Bielefeld-CeBiTec--protocol_heatshock.pdf">transformation via heat shock</a></li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Cellgrowth with the plasmid </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Christina Drake</p> | ||
+ | <p><b>Superior experiment:</b> Overnightculture of tRNA-psB1C3</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> 3 ml <a target="blank" href="https://static.igem.org/mediawiki/2017/a/a7/T--Bielefeld-CeBiTec--protocol_LB.pdf">LB-medium</a> and 0,75 µl chloramphenicol</li> | ||
+ | <li> inkubation overnight 37°C, 140rpm</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Cloning of the ori (pSB6A1) in pSB1C3 </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Laura Schlueter</p> | ||
+ | <p><b>Superior experiment:</b> aaRS Plasmid (without aaRS)</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> <a target="blank" href="https://static.igem.org/mediawiki/2017/5/55/T--Bielefeld-CeBiTec--protocol_gibson_assembly.pdf">Gibson Assembly</a> Master Mix</li> | ||
+ | <ul> | ||
+ | <li> Backbone: 3.28 µL Ori 1.0: 1.73 µL</li> | ||
+ | <li> Backbone: 0.32 µL Ori 1.1: 4.68 µL</li> | ||
+ | <li> Backbone: 0.90 µL Ori 3.0: 4.10 µL</li> | ||
+ | </ul> | ||
+ | <li> Trafo: heatshock</li> | ||
+ | <li> not successfull because of the wrong overlapps (try again)</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Colony PCR of the Retrafo of pRS (because of mixed culture) </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Laura Schlueter</p> | ||
+ | <p><b>Superior experiment:</b> aaRS plasmid</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> Sequencing was not succesfull (maybe mixed colony), so I did a retrafo with the aim of a successful sequencing (incorporation of the aaRS in pSB1C3)</li> | ||
+ | <ul> | ||
+ | <li> pRS 3: 1-5</li> | ||
+ | <li> pRS 4: 1-5</li> | ||
+ | <li> pRS 5: 1-5</li> | ||
+ | <li> pRS 7: 1-5</li> | ||
+ | <li> no positive result (no band at 1000 bp)</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Amplification of pSB1C3 for the cloning of the ori in pSB1C3 </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Laura Schlueter</p> | ||
+ | <p><b>Superior experiment:</b> aaRS plasmid</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> <a target="blank" href="https://static.igem.org/mediawiki/2017/8/88/T--Bielefeld-CeBiTec--protocol_PCR_Q5_HF_Polymerase.pdf">PCR with Q5 High-Fidelity Polymerase</a> Master Mix, Primer jb,jn, 65°C</li> | ||
+ | <li> gelelectrophoresis, 1% Agarose</li> | ||
+ | <li> band 2000-2500 bp (supposed to be around 2200 bp)</li> | ||
+ | <li> Clean up from the gel</li> | ||
+ | <ul> | ||
+ | <li> pSB1C3(nur ori): 22.2 ng/µL</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Amplification of pSB1C3 fragments for a 4 part Gibson (see page 4 in the paper-labbook) </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Laura Schlueter</p> | ||
+ | <p><b>Superior experiment:</b> aaRS plasmid</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> PCR, Q5 Master Mix</li> | ||
+ | <ul> | ||
+ | <li> Primer jj, jn for fragment 2, 65°C</li> | ||
+ | <li> Primer jb, ht for fragment 4, 65°C</li> | ||
+ | </ul> | ||
+ | <li> gelelectrophoresis, 1% Agarose</li> | ||
+ | <ul> | ||
+ | <li> band at 300 (fragment 2, supposed to be around 326 bp)</li> | ||
+ | <li> band at 1200 (fragment 4, supposed to be around 1233 bp)</li> | ||
+ | </ul> | ||
+ | <li> Clean up from the gel</li> | ||
+ | <ul> | ||
+ | <li> fragment 2: 18.5 ng/µL (overlapp(aaRS)_pSB1C3_overlapp(ori))</li> | ||
+ | <li> fragment 4: 19.1 ng/µL (overlapp(ori)_pSB1C3_overlapp(aaRS))</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Colony PCR of 2B2-2B8, B1 and A1 </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Maximilian Edich</p> | ||
+ | <p><b>Superior experiment:</b> Biobrick Construction with RuBisCo Parts</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> <a target="blank" href="https://static.igem.org/mediawiki/2017/c/cc/T--Bielefeld-CeBiTec--protocol_colonyPCR_GoTaq_G2.pdf">GoTaq® G2 PCR</a></li> | ||
+ | <li> most have negative results -> create new destination plasmid pSB1A3</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Colony PCR of retrafo and more colonies of the pRS </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Laura Schlueter</p> | ||
+ | <p><b>Superior experiment:</b> aaRS plasmid</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> GoTaq Master Mix, Primer vr,vf</li> | ||
+ | <ul> | ||
+ | <li> 2 strong bands at 400 bp and 600 bp, small bands of pRS3 6 and pRS4 9 at around 1200-1500 bp</li> | ||
+ | <li> bands of pRS31, pRS32, pRS34, pRS36, pRS39 at around 1200-1500 bp</li> | ||
+ | </ul> | ||
+ | <li> GoTaq Master Mix, Primer hq, jk (<a target="blank" href="https://static.igem.org/mediawiki/2017/5/55/T--Bielefeld-CeBiTec--protocol_gibson_assembly.pdf">Gibson Assembly</a> primer) with the colonies pRS31, pRS32, pRS34, pRS36, pRS39</li> | ||
+ | <ul> | ||
+ | <li> pRS31, pRS32, pRS39 : strong bands at around 1200 bp</li> | ||
+ | </ul> | ||
+ | <li> the two positive colony PCRs (vf-vr, hq-jk) for these probes, successful integration of the Tyr-aaRS in pSB1C3 seems to be successfull</li> | ||
+ | <li> pRS31, pRS32, pRS39 send to sequencing</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> pRS31, pRS32, pRS39 plasmid isolation </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Laura Schlueter</p> | ||
+ | <p><b>Superior experiment:</b> aaRS plasmid</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> Macherey-Nagel purification kit</li> | ||
+ | <ul> | ||
+ | <li> pRS31: 324.1 ng/µL</li> | ||
+ | <li> pRS32: 231.8 ng/µL</li> | ||
+ | <li> pRS39: 225.5 ng/µL</li> | ||
+ | </ul> | ||
+ | <li> send tosequencing</li> | ||
+ | <li> pRS31, pRS39 have the pSB1C3 with the Tyr-aaRS incorporated (but on position 32, the Arginine (cgt) should be changed to an Leucine (ctg), but it is not. On position 290 there really is a missing Leu, on position 268 the Asparagin is changed to an Arginine)</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-date"> | ||
+ | <h3> 2017-07-31 - 2017-08-06 </h3> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> construct a biobrick </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Christina Drake</p> | ||
+ | <p><b>Superior experiment:</b> Aquacloning of tRNA and backbone</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> transformation via heatschock</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> plasmid isolation of the positive colonies of the retrafo pRS4 4 (wrong!) and pRS3 12 </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Laura Schlueter</p> | ||
+ | <p><b>Superior experiment:</b> aaRS plasmid</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> Macherey-Nagel purification kit with each 3 mL culture</li> | ||
+ | <ul> | ||
+ | <li> pRS4 4: 44 ng/µL</li> | ||
+ | <li> pRS3 12: 68.3 ng/µL</li> | ||
+ | <li> not send to sequencing jet</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> 4 part Gibson and Trafo </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Laura Schlueter</p> | ||
+ | <p><b>Superior experiment:</b> aaRS plasmid</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> <a target="blank" href="https://static.igem.org/mediawiki/2017/5/55/T--Bielefeld-CeBiTec--protocol_gibson_assembly.pdf">Gibson Assembly</a> Master Mix with 5 µl template</li> | ||
+ | <ul> | ||
+ | <li> pSB1C3 fragment (2): 18.5 ng/µL around 350 bp 2.2 µL</li> | ||
+ | <li> pSB1C3 fragment (4): 19.1 ng/µL around 1300 bp 0.8 µL</li> | ||
+ | <li> oK1.1 (pMB1 from pSB6A1): 5.8 ng/µL around 1300 bp 1.8 µL</li> | ||
+ | <li> Tyr-aaRS: 68.5 ng/µL around 1000 bp 0.2 µL</li> | ||
+ | </ul> | ||
+ | <li> Trafo via heatshock</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Gibson of the ori in pSB1C3 (pori) and Trafo </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Laura Schlueter</p> | ||
+ | <p><b>Superior experiment:</b> aaRS plasmid</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> <a target="blank" href="https://static.igem.org/mediawiki/2017/5/55/T--Bielefeld-CeBiTec--protocol_gibson_assembly.pdf">Gibson Assembly</a> Master Mix with 5 µL template</li> | ||
+ | <ul> | ||
+ | <li> pSB1C3(nur ori): 22.2 ng/µL around 2100 bp 1.57 µL</li> | ||
+ | <li> ok1.0: 5.80 ng/µL around 1258 bp 3.43 µL</li> | ||
+ | </ul> | ||
+ | <li> Trafo via heatshock</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Transfer positive colonies </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Maximilian Edich</p> | ||
+ | <p><b>Superior experiment:</b> Biobrick Construction with RuBisCo Parts</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> picked and transfered positiv colonies of 2B2, 2B6, 2B7, 2B8 and A1 onto a new plate</li> | ||
+ | <li> incubate over night</li> | ||
+ | <li> 2B2 and 2B8 are contaminated with negativ colonies</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> construct a biobrick </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Christina Drake</p> | ||
+ | <p><b>Superior experiment:</b> Aquacloning of tRNA and backbone</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> transformation via heatschock</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> construct a biobrick </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Christina Drake</p> | ||
+ | <p><b>Superior experiment:</b> Gibbsonassembly of barnase and backbone</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> transformation via heatschock</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Preculture of 2B6, 2B7 and A1 </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Maximilian Edich</p> | ||
+ | <p><b>Superior experiment:</b> Biobrick Construction with RuBisCo Parts</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> used 3µl <a target="blank" href="https://static.igem.org/mediawiki/2017/a/a7/T--Bielefeld-CeBiTec--protocol_LB.pdf">LB media</a></li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Cellgrowth with the plasmid </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Christina Drake</p> | ||
+ | <p><b>Superior experiment:</b> Overnightculture of tRNA-psB1C3 and barnase-psB1C3</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> 3 ml <a target="blank" href="https://static.igem.org/mediawiki/2017/a/a7/T--Bielefeld-CeBiTec--protocol_LB.pdf">LB-medium</a> and 0,75 µl chloramphenicol</li> | ||
+ | <li> inkubation overnight 37°C, 140rpm</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Plasmidisolation </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Maximilian Edich</p> | ||
+ | <p><b>Superior experiment:</b> Biobrick Construction with RuBisCo Parts</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> Nucleospin Plasmidisolation protocol</li> | ||
+ | <ul> | ||
+ | <li> got 67.3 ng/µl of 2B6</li> | ||
+ | <li> got 32.7 ng/µl of 2B7</li> | ||
+ | <li> got 51.6 ng/µl of A1</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> To get and purified the plasmid </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Christina Drake</p> | ||
+ | <p><b>Superior experiment:</b> Plasmidisolation of barnase-plasmid and tRNA-plasmids</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> protocol from Macherley-Nagel: Plasmid DNA purification, protocol 5: NucleoSpin</li> | ||
+ | <li> concentration measurement by nanoprop</li> | ||
+ | <li> sequenzingorder</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Redo digestion of the destination plasmid </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Maximilian Edich</p> | ||
+ | <p><b>Superior experiment:</b> Biobrick Construction with RuBisCo Parts</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> analyze the destination plasmid digestion via gelelectrophoresis</li> | ||
+ | <ul> | ||
+ | <li> pSB1A3 digestion was not effective</li> | ||
+ | </ul> | ||
+ | <li> redo the digestion and analyze it on the gel</li> | ||
+ | <ul> | ||
+ | <li> new dgestion was very effective</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-date"> | ||
+ | <h3> 2017-08-07 - 2017-08-13 </h3> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Amplification of the backbone (pSB1C3) and insert (Tyr-aaRS) </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Laura Schlueter</p> | ||
+ | <p><b>Superior experiment:</b> aaRS plasmid</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> PCR Q5 Polymerase (no Master Mix)</li> | ||
+ | <ul> | ||
+ | <li> for 50 µL reaction</li> | ||
+ | <ul> | ||
+ | <li> 10 µL Reaction Buffer</li> | ||
+ | <li> 5 µL 2mM dNTPs</li> | ||
+ | <li> 1 µL Template</li> | ||
+ | <li> 0,5 µL High Fidelity DNA Polymerase</li> | ||
+ | <li> 10.0 µL High GC Enhancer</li> | ||
+ | <li> 18.5 µL ddest H2O</li> | ||
+ | <li> 5.0 µL Primer</li> | ||
+ | <ul> | ||
+ | <li> pSB1C3 (from RuBisCo, ncAA team): ht, jk 65°C</li> | ||
+ | <li> aaRS (from Heidelberg) : hq, jk 65°C (mistake! should be 58°C, redone identically with 58°C)</li> | ||
+ | </ul> | ||
+ | <li> digestion of the PCR products with Dpn1: 5 µL CutSmart Reaktionsbuffer and 1 µL Dpn1 per 50 µL PCR product, on 37°C over night)</li> | ||
+ | </ul> | ||
+ | <li> agarose-gelelectrophhoresis, 1% (bands of the right size)</li> | ||
+ | </ul> | ||
+ | <li> No further use, because results of the sequencing which show that the integration of the incorporation of the Tyr-aaRS in pSB1C3 was already successful (30.06.2017)</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Ligation of 2B2-2B5 and B1 </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Maximilian Edich</p> | ||
+ | <p><b>Superior experiment:</b> Biobrick Construction with RuBisCo Parts</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> Ligation protocol from the <a target="blank" href="https://static.igem.org/mediawiki/2017/8/84/T--Bielefeld-CeBiTec--protocol_Standard_Biobrick.pdf">BioBrick Assembly</a></li> | ||
+ | <ul> | ||
+ | <li> Variation: incubated 1h at RT instead of 10min</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> characterisation of T7GFP revers </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Christina Drake</p> | ||
+ | <p><b>Superior experiment:</b> preparation of KRX-competent cells</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> heatschocktransformation of T7GFP and T7GFP revers in KRX</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> construct a biobrick for the selectionplasmid </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Christina Drake</p> | ||
+ | <p><b>Superior experiment:</b> PCR of T7RNA-Polymerase</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> 1%-agarose-<a target="blank" href="https://static.igem.org/mediawiki/2017/d/d4/T--Bielefeld-CeBiTec--protocol_TAE_buffer.pdf">TAE</a>-gel</li> | ||
+ | <li> result: PCR did work</li> | ||
+ | <li> <a target="blank" href="https://static.igem.org/mediawiki/2017/5/55/T--Bielefeld-CeBiTec--protocol_gibson_assembly.pdf">Gibson Assembly</a></li> | ||
+ | <li> <a target="blank" href="https://static.igem.org/mediawiki/2017/a/a0/T--Bielefeld-CeBiTec--protocol_heatshock.pdf">transformation via heat shock</a></li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Amplification of the backbone (pRS31) and insert (ori from pSB6A1, oK1.0) for Gibson assembly </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Laura Schlueter</p> | ||
+ | <p><b>Superior experiment:</b> aaRS plasmid</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> PCR Q5 Polymerase (protocoll of 07.08.2017)</li> | ||
+ | <ul> | ||
+ | <li> pRS31: primer jb, jn 65°C</li> | ||
+ | <li> oK1: primer jl, jc 65°C</li> | ||
+ | </ul> | ||
+ | <li> digestion with Dpn1 (protocoll of 07.08.2017)</li> | ||
+ | <li> agarose-gelelectrophhoresis, 1%</li> | ||
+ | <ul> | ||
+ | <li> band around 1200 bp (oK1.1) / 2500 bp (pRS31), positive</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Gibson Assembly and Trafo of the pRS31 and oK1.0 </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Laura Schlueter</p> | ||
+ | <p><b>Superior experiment:</b> aaRS plasmid</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> agarose-gelelectrophhoresis, 1%</li> | ||
+ | <li> purification from the gel with the Macherey-Nagel purification kit</li> | ||
+ | <ul> | ||
+ | <li> oK1.1: 22.5 ng/µL</li> | ||
+ | <li> pRS32: 9.5 ng/µL</li> | ||
+ | </ul> | ||
+ | <li> <a target="blank" href="https://static.igem.org/mediawiki/2017/5/55/T--Bielefeld-CeBiTec--protocol_gibson_assembly.pdf">Gibson Assembly</a> Master Mix with:</li> | ||
+ | <ul> | ||
+ | <li> 4.1 µL of oK1.1: around 2480 bp 9.5 ng/µL</li> | ||
+ | <li> 0.9 µL of pRS32: around 1250 bp 22.5 ng/µL</li> | ||
+ | </ul> | ||
+ | <li> Trafo via heatshock</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> getting positive clones for further work </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Yannic Kerkhoff</p> | ||
+ | <p><b>Superior experiment:</b> heatshock transformation of assembled fragments F13 and NPA-RS gene synthesis</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> standard heatshock protocol</li> | ||
+ | <ul> | ||
+ | <li> used own produced chemo competend DH5alpha</li> | ||
+ | <li> streaked out on LB-plates with Cam</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> multiplication, verification and purification of the needed fragment F13: linearized ONBY-Part with removal of the ONBY-RS </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Yannic Kerkhoff</p> | ||
+ | <p><b>Superior experiment:</b> Q5-PCR, gel and PCR-cleanUp of F13</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> standard Q5-PCR protocol</li> | ||
+ | <ul> | ||
+ | <li> template: K1416000</li> | ||
+ | <li> primer fwd: 17hl</li> | ||
+ | <li> primer rev: 17mv</li> | ||
+ | <li> annealing temperature: 63°C</li> | ||
+ | <li> extension time: 90 seconds</li> | ||
+ | </ul> | ||
+ | <li> standard PCR-cleanUp protocol</li> | ||
+ | <ul> | ||
+ | <li> 80,6 ng/µL</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Assemble the fragments to get the Part BBa_K2201200 </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Yannic Kerkhoff</p> | ||
+ | <p><b>Superior experiment:</b> Gibson assembly of F13 and NPA-RS gene synthesis</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> standard <a target="blank" href="https://static.igem.org/mediawiki/2017/5/55/T--Bielefeld-CeBiTec--protocol_gibson_assembly.pdf">Gibson Assembly</a> protocol</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> getting positive clones for further work </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Yannic Kerkhoff</p> | ||
+ | <p><b>Superior experiment:</b> heatshock transformation of assembled fragments F5,F1,F3</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> standard heatshock protocol</li> | ||
+ | <ul> | ||
+ | <li> used own produced chemo competend DH5alpha</li> | ||
+ | <li> streaked out on LB-plates with Cam</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> multiplication, verification and purification of the needed fragment F1: GFPLinker1 </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Yannic Kerkhoff</p> | ||
+ | <p><b>Superior experiment:</b> Q5-PCR, gel and PCR-cleanUp of F1</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> standard Q5-PCR protocol</li> | ||
+ | <ul> | ||
+ | <li> template: BBa_E0400</li> | ||
+ | <li> primer fwd: 17gk</li> | ||
+ | <li> primer rev: 17gm</li> | ||
+ | <li> annealing temperature: 58°C</li> | ||
+ | <li> extension time: 50 seconds</li> | ||
+ | </ul> | ||
+ | <li> standard PCR-cleanUp protocol</li> | ||
+ | <ul> | ||
+ | <li> 15,9 ng/µL</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> multiplication, verification and purification of the needed fragment F3: StrepLinker1 </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Yannic Kerkhoff</p> | ||
+ | <p><b>Superior experiment:</b> Q5-PCR, gel and PCR-cleanUp of F3</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> standard Q5-PCR protocol</li> | ||
+ | <ul> | ||
+ | <li> template: BBa_KJ36848</li> | ||
+ | <li> primer fwd: 17go</li> | ||
+ | <li> primer rev: 17gq</li> | ||
+ | <li> annealing temperature: 61°C</li> | ||
+ | <li> extension time: 30 seconds</li> | ||
+ | </ul> | ||
+ | <li> standard PCR-cleanUp protocol</li> | ||
+ | <ul> | ||
+ | <li> 17,9 ng/µL</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Biobrickassembly </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Maximilian Edich</p> | ||
+ | <p><b>Superior experiment:</b> Biobrick Construction with RuBisCo Parts</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> <a target="blank" href="https://static.igem.org/mediawiki/2017/8/84/T--Bielefeld-CeBiTec--protocol_Standard_Biobrick.pdf">BioBrick Assembly</a></li> | ||
+ | <li> repeat digestion of T7-Terminator downstrampart</li> | ||
+ | <li> ligate over night</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Transformation of new plasmids </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Maximilian Edich</p> | ||
+ | <p><b>Superior experiment:</b> Biobrick Construction with RuBisCo Parts</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> <a target="blank" href="https://static.igem.org/mediawiki/2017/a/a0/T--Bielefeld-CeBiTec--protocol_heatshock.pdf">transformation via heat shock</a></li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> multiplication, verification and purification of the needed fragment F15: linear GLS1 </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Yannic Kerkhoff</p> | ||
+ | <p><b>Superior experiment:</b> Q5-PCR, gel and PCR-cleanUp of F15</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> standard Q5-PCR protocol</li> | ||
+ | <ul> | ||
+ | <li> template: P2: GLS2</li> | ||
+ | <li> primer fwd: 17go</li> | ||
+ | <li> primer rev: 17gm</li> | ||
+ | <li> annealing temperature: 60°C</li> | ||
+ | <li> extension time: 190 seconds</li> | ||
+ | </ul> | ||
+ | <li> standard PCR-cleanUp protocol</li> | ||
+ | <ul> | ||
+ | <li> 36,9 ng/µL</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Primer annealing preparation </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Camilla Maerz</p> | ||
+ | <p><b>Superior experiment:</b> M.A.X restriction system - Primer annealing test</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> Both Primers (17jh; 17ji) were resuspended in 1x Composite Buffer</li> | ||
+ | <li> Dilutions: 100 µM, 10 µM, 5 µM, 2.5 µM, 0.5 µM</li> | ||
+ | <li> OD 260 were measured to determine the real concentration</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-date"> | ||
+ | <h3> 2017-08-14 - 2017-08-20 </h3> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Screening for positive transformations containing the Biobrick </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Yannic Kerkhoff</p> | ||
+ | <p><b>Superior experiment:</b> GoTaq PCR of 4 clones of BBa_K2201220</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> standard GoTaq-protocol</li> | ||
+ | <ul> | ||
+ | <li> template: colonie pcr clones of GA_K2201220</li> | ||
+ | <li> primer fwd: Präfix_fwd</li> | ||
+ | <li> primer rev: Suffix_rev</li> | ||
+ | <li> annealing temperature: 56°C</li> | ||
+ | <li> extension time: 40 seconds</li> | ||
+ | </ul> | ||
+ | <li> positive clone 3</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> multiplication of plasmids containing the Biobrick </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Yannic Kerkhoff</p> | ||
+ | <p><b>Superior experiment:</b> preculture of the positive clone 3 of BBa_K2201220</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> standard procedure</li> | ||
+ | <ul> | ||
+ | <li> 1,25µl Cam in 5mL LB</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Primer annealing of 17jh & 17ji </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Camilla Maerz</p> | ||
+ | <p><b>Superior experiment:</b> M.A.X restriction system - Primer annealing test</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> Protocol for annealing oligonucleotides (sigmaaldrich)</li> | ||
+ | <ul> | ||
+ | <li> Eqimolar Primers were mixed (50 µL)</li> | ||
+ | <li> Thermal profile (thermocycler)</li> | ||
+ | <ul> | ||
+ | <li> 95 °C, 2 min</li> | ||
+ | <li> Cool to 25 °C over 45 min</li> | ||
+ | <li> Cool to 4 °C for temporary storage</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Integration of barnase in pSB1C3 </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Olga Schmidt</p> | ||
+ | <p><b>Superior experiment:</b> Construction of selection plasmid</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> <a target="blank" href="https://static.igem.org/mediawiki/2017/2/23/T--Bielefeld-CeBiTec--protocol_Restriction_Digest.pdf">Restriction Digest</a> of pSB1C3_I749606</li> | ||
+ | <ul> | ||
+ | <li> 5 µL 10x Universal buffer</li> | ||
+ | <li> 5.54 µL plasmid</li> | ||
+ | <li> 1µL <i>Xba</i>I</li> | ||
+ | <li> 1µL <i>Pst</i>I</li> | ||
+ | <li> filled up to final volume 50 µL with ddH20</li> | ||
+ | <li> Incubation: 30 min, 37 °C</li> | ||
+ | <li> Heatinactivation: 20 min, 80 °C</li> | ||
+ | </ul> | ||
+ | <li> Agarose gel electrophoresis (1%)</li> | ||
+ | <ul> | ||
+ | <li> 100 V, 30 min</li> | ||
+ | </ul> | ||
+ | <li> PCR and DNA purification kit (Macherey-Nagel)</li> | ||
+ | <ul> | ||
+ | <li> Nucleo spin PCR and <a target="blank" href="https://static.igem.org/mediawiki/2017/0/0e/T--Bielefeld-CeBiTec--protocol_Monarch_PCR_and_DNA_clean_up_kit_NEB.pdf">Monarch® PCR & DNA Clean-up</a> protocol</li> | ||
+ | </ul> | ||
+ | <li> <a target="blank" href="https://static.igem.org/mediawiki/2017/5/55/T--Bielefeld-CeBiTec--protocol_gibson_assembly.pdf">Gibson Assembly</a> protocol</li> | ||
+ | <li> <a target="blank" href="https://static.igem.org/mediawiki/2017/a/a0/T--Bielefeld-CeBiTec--protocol_heatshock.pdf">transformation via heat shock</a> protocol in <i>E. coli</i> DH5<i>alpha</i></li> | ||
+ | <li> <a target="blank" href="https://static.igem.org/mediawiki/2017/a/ab/T--Bielefeld-CeBiTec--protocol_electrotrafo.pdf">transformation via electroporation</a> protocol in <i>E. coli</i> DH5<i>alpha</i></li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> PCR of T7RNAP_BL </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Olga Schmidt</p> | ||
+ | <p><b>Superior experiment:</b> Construction of the selection plasmid</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> PCR</li> | ||
+ | <ul> | ||
+ | <li> Go Tag (Promega) protocol</li> | ||
+ | <li> Danaturation: 56 °C</li> | ||
+ | <li> Elongation: 180 s</li> | ||
+ | </ul> | ||
+ | <li> Agarose gel electrophoresis (1%)</li> | ||
+ | <ul> | ||
+ | <li> 100 V, 30 min</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> getting plasmids containing the Biobrick for further experiments </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Yannic Kerkhoff</p> | ||
+ | <p><b>Superior experiment:</b> plasmid isolation of preculture of BBa_K2201220 clone 3</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> standard protocol</li> | ||
+ | <ul> | ||
+ | <li> 108,9 ng/µL</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Combination of two biobricks to get the composite part BBa_K2201320 for protein expression </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Yannic Kerkhoff</p> | ||
+ | <p><b>Superior experiment:</b> Biobrick Assembly of BBa_K608006 and BBa_K2201220</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> <a target="blank" href="https://static.igem.org/mediawiki/2017/8/84/T--Bielefeld-CeBiTec--protocol_Standard_Biobrick.pdf">Standard BioBrick Assembly</a> protocol</li> | ||
+ | <ul> | ||
+ | <li> Backbone: BBa_J04450, 7,7µL</li> | ||
+ | <li> upstream: BBa_K608006, 3,6µL</li> | ||
+ | <li> downstram: BBa_K2201220, 5,0µL</li> | ||
+ | <li> Ligation time was 12 h at 8 °C</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Colony and plasmid PCR of T7RNAP_BL c1-4 </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Olga Schmidt</p> | ||
+ | <p><b>Superior experiment:</b> Construction of the selection plasmid</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> PCR</li> | ||
+ | <ul> | ||
+ | <li> Go Tag (Promega) protocol</li> | ||
+ | <li> Primer: VR & VF</li> | ||
+ | <li> Danaturation: 56 °C</li> | ||
+ | <li> Elongation: 180 s</li> | ||
+ | </ul> | ||
+ | <li> Agarose gel electrophoresis (1%)</li> | ||
+ | <ul> | ||
+ | <li> 100 V, 30 min</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> getting positive clones for further work </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Yannic Kerkhoff</p> | ||
+ | <p><b>Superior experiment:</b> heatshock transformation of the Biobrick Assembly product BBa_K2201320</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> standard heatshock protocol</li> | ||
+ | <ul> | ||
+ | <li> used own produced chemo competend BL21(DE3)</li> | ||
+ | <li> streaked out on LB-plates with Cam</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Colony PCR of pSB1C3_barnase (gblock) c1-70 </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Olga Schmidt</p> | ||
+ | <p><b>Superior experiment:</b> Construction of selection plasmid</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> PCR</li> | ||
+ | <ul> | ||
+ | <li> Go Tag (Promega) protocol</li> | ||
+ | <li> Primer: Vr & VF2</li> | ||
+ | <li> Danaturation: 56.5 °C</li> | ||
+ | <li> Elongation: 40 s</li> | ||
+ | </ul> | ||
+ | <li> Agarose gel electrophoresis (1%)</li> | ||
+ | <ul> | ||
+ | <li> 100 V, 30 min</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Screening for positive transformations containing the Biobrick </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Yannic Kerkhoff</p> | ||
+ | <p><b>Superior experiment:</b> GoTaq PCR of 19 clones of BBa_K2201320</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> standard GoTaq-protocol</li> | ||
+ | <ul> | ||
+ | <li> template: colonie pcr clones of GA_K2201320</li> | ||
+ | <li> primer fwd: T7-eva_fwd</li> | ||
+ | <li> primer rev: Suffix_rev</li> | ||
+ | <li> annealing temperature: 51°C</li> | ||
+ | <li> extension time: 80 seconds</li> | ||
+ | </ul> | ||
+ | <li> positive clone 19</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> multiplication of plasmids containing the Biobrick </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Yannic Kerkhoff</p> | ||
+ | <p><b>Superior experiment:</b> preculture of the positive clone 19 of BBa_K2201320</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> standard procedure</li> | ||
+ | <ul> | ||
+ | <li> 1,25µl Cam in 5mL LB</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Combination of two biobricks to get the composite part BBa_K2201321 for protein expression </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Yannic Kerkhoff</p> | ||
+ | <p><b>Superior experiment:</b> Biobrick Assembly of BBa_K608006 and BBa_K2201221</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> <a target="blank" href="https://static.igem.org/mediawiki/2017/8/84/T--Bielefeld-CeBiTec--protocol_Standard_Biobrick.pdf">Standard BioBrick Assembly</a> protocol</li> | ||
+ | <ul> | ||
+ | <li> Backbone: BBa_J04450, 2,0µL</li> | ||
+ | <li> upstream: BBa_K608006, 3,6µL</li> | ||
+ | <li> downstram: BBa_K2201221, 2,4µL</li> | ||
+ | <li> Ligation time was 12 h at 8°C</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Primer annealing of 17jl & 17jm </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Camilla Maerz</p> | ||
+ | <p><b>Superior experiment:</b> M.A.X restriction system - Primer annealing test</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> Both Primers were resuspended in ddH20 to a final concentration of 100 µM</li> | ||
+ | <li> HEPES annealing standard protocol</li> | ||
+ | <li> Aqua annealing standard protocol</li> | ||
+ | <ul> | ||
+ | <li> Final volume: 20 µL</li> | ||
+ | </ul> | ||
+ | <li> Agarose gel electrophoresis (2%)</li> | ||
+ | <ul> | ||
+ | <li> 60 V, 50 min</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> multiplication of plasmids containing the Biobrick </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Yannic Kerkhoff</p> | ||
+ | <p><b>Superior experiment:</b> preculture of BL21(DE3) BBa_K2201320</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> standard procedure</li> | ||
+ | <ul> | ||
+ | <li> 1,25µl Cam in 10mL LB</li> | ||
+ | <li> 12 h at 37°C</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> expression of the fusion protein </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Yannic Kerkhoff</p> | ||
+ | <p><b>Superior experiment:</b> preculture of BL21(DE3) BBa_K2201320 with IPTG</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> standard procedure</li> | ||
+ | <ul> | ||
+ | <li> 1,25µl Cam in 10mL LB</li> | ||
+ | <li> 12 h at 37°C</li> | ||
+ | <li> induced with IPTG 0.8mM</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> getting plasmids containing the Biobrick for further experiments </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Yannic Kerkhoff</p> | ||
+ | <p><b>Superior experiment:</b> plasmid isolation of preculture of BBa_K2201320 clone 19</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> standard protocol</li> | ||
+ | <ul> | ||
+ | <li> 454,4 ng/µL</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> multiplication of plasmids containing the Biobrick </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Yannic Kerkhoff</p> | ||
+ | <p><b>Superior experiment:</b> preculture of BL21(DE3) BBa_K2201320</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> standard procedure</li> | ||
+ | <ul> | ||
+ | <li> 1,25µl Cam in 10mL LB</li> | ||
+ | <li> 12 h at 37°C</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> expression of the fusion protein </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Yannic Kerkhoff</p> | ||
+ | <p><b>Superior experiment:</b> preculture of BL21(DE3) BBa_K2201320 with IPTG</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> standard procedure</li> | ||
+ | <ul> | ||
+ | <li> 1,25µl Cam in 10mL LB</li> | ||
+ | <li> 12 h at 37°C</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Ligation of pSB1C3 and barnase (gblock) </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Olga Schmidt</p> | ||
+ | <p><b>Superior experiment:</b> Construction of selection plasmid</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> Ligation reaction</li> | ||
+ | <ul> | ||
+ | <li> 7 µL vector</li> | ||
+ | <li> 3 µL gblock</li> | ||
+ | <li> 2 µL T4 DNA ligase buffer</li> | ||
+ | <li> 1 µL T4 DNA ligase</li> | ||
+ | <li> 17 µL ddH20</li> | ||
+ | </ul> | ||
+ | <li> Incubation: RT, 1 h</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Colony PCR of pSB1C3_barnaseBL c1-60 </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Olga Schmidt</p> | ||
+ | <p><b>Superior experiment:</b> Construction of selection plasmid</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> PCR</li> | ||
+ | <ul> | ||
+ | <li> Q5 High-fidelily (NEB) protocol</li> | ||
+ | <li> Denaturation: 56.5 °C</li> | ||
+ | <li> Elongation: 45 s</li> | ||
+ | </ul> | ||
+ | <li> Agarose gel electrophoresis (1%)</li> | ||
+ | <ul> | ||
+ | <li> 100 V, 30 min</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> getting positive clones for further work </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Yannic Kerkhoff</p> | ||
+ | <p><b>Superior experiment:</b> heatshock transformation of the Biobrick Assembly product BBa_K2201321</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> standard heatshock protocol</li> | ||
+ | <ul> | ||
+ | <li> used own produced chemo competend BL21(DE3)</li> | ||
+ | <li> streaked out on LB-plates with Cam</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> - standard cultivation protocoll </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Yannic Kerkhoff</p> | ||
+ | <p><b>Superior experiment:</b> Cultivation of BL21(DE3) with BBa_K2201320</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> 2x 100mL <a target="blank" href="https://static.igem.org/mediawiki/2017/a/a7/T--Bielefeld-CeBiTec--protocol_LB.pdf">LB-media</a> with 25µL Cam in 1000mL flasks</li> | ||
+ | <ul> | ||
+ | <li> innoculation with 3.3 mL preculture of OD 3 to get a starting OD of 0.1</li> | ||
+ | <li> incubation for four hours to a OD of 0.6</li> | ||
+ | <li> addition of 500µL 0.5mM IPTG each</li> | ||
+ | <li> incubation for 8 hours at 37°C and 24 hours at 18°C</li> | ||
+ | <li> harveting of the cells</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Native PAGE of annealed primers (17jl & 17jm) </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Camilla Maerz</p> | ||
+ | <p><b>Superior experiment:</b> M.A.X restriction system - Primer annealing test</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> <a target="blank" href="https://static.igem.org/mediawiki/2017/e/e6/T--Bielefeld-CeBiTec--protocol_native_PAGE.pdf">Native DNA PAGE</a> standard protocol</li> | ||
+ | <ul> | ||
+ | <li> tested samples:</li> | ||
+ | <ul> | ||
+ | <li> HEPES annealing (17jl & 17jm): 100 µM, 10 µM, 5 µM, 2.5 µM, 1 µM, 0.5 µM</li> | ||
+ | <li> Aqua annealing (17jl & 17jm): 100 µM, 10 µM, 5 µM, 2.5 µM, 1 µM, 0.5 µM</li> | ||
+ | </ul> | ||
+ | <li> Variation: ~40 mA</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-date"> | ||
+ | <h3> 2017-08-21 - 2017-08-27 </h3> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Native PAGE of annealed primers (17jl & 17jm) </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Camilla Maerz</p> | ||
+ | <p><b>Superior experiment:</b> M.A.X restriction system - Primer annealing test</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> <a target="blank" href="https://static.igem.org/mediawiki/2017/e/e6/T--Bielefeld-CeBiTec--protocol_native_PAGE.pdf">Native DNA PAGE</a> standard protocol</li> | ||
+ | <ul> | ||
+ | <li> tested samples:</li> | ||
+ | <ul> | ||
+ | <li> HEPES annealing (17jl & 17jm): 100 µM, 10 µM, 5 µM, 2.5 µM, 1 µM, 0.5 µM</li> | ||
+ | <li> Aqua annealing (17jl & 17jm): 100 µM, 10 µM, 5 µM, 2.5 µM, 1 µM, 0.5 µM</li> | ||
+ | </ul> | ||
+ | <li> Variation: 100 V, 1 h</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Plasmidisolation of pSB1C3_barnaseB c5, 22, 30, 31, 32, 45 </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Olga Schmidt</p> | ||
+ | <p><b>Superior experiment:</b> Construction of selection plasmid</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> Plasmid DNA purification kit (Zymo PURE)</li> | ||
+ | <ul> | ||
+ | <li> Nucleo spin plasmid protocol</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> PCR of KanR & pSB1C3 </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Olga Schmidt</p> | ||
+ | <p><b>Superior experiment:</b> Construction of selection plasmid</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> PCR</li> | ||
+ | <ul> | ||
+ | <li> Q5 High-fidelily (NEB) protocol</li> | ||
+ | <li> Primer: 17nx & 17ny (Kan)</li> | ||
+ | <li> Primer: 17od & 17oe (pSB1C3)</li> | ||
+ | <li> Denaturation: 54 °C & 60 °C</li> | ||
+ | <li> Elongation: 30 s & 75 s</li> | ||
+ | </ul> | ||
+ | <li> <a target="blank" href="https://static.igem.org/mediawiki/2017/2/23/T--Bielefeld-CeBiTec--protocol_Restriction_Digest.pdf">Restriction Digest</a></li> | ||
+ | <ul> | ||
+ | <li> 5 µL 10x CutSmart buffer</li> | ||
+ | <li> 1 µg plasmid</li> | ||
+ | <li> 1 µL <i>Dpn</i>I</li> | ||
+ | <li> filled up to final volume 50 µL with ddH20</li> | ||
+ | <li> Incubation: ON, 37 °C</li> | ||
+ | <li> Inactivation: 20 min, 80 °C</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> multiplication of plasmids containing the Biobrick </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Yannic Kerkhoff</p> | ||
+ | <p><b>Superior experiment:</b> preculture of the positive clone 3 of BBa_K2201200</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> standard procedure</li> | ||
+ | <ul> | ||
+ | <li> 1,25µl Cam in 5mL LB</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Restriction digest of KanR fragment </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Olga Schmidt</p> | ||
+ | <p><b>Superior experiment:</b> Construction of selection plasmid</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> <a target="blank" href="https://static.igem.org/mediawiki/2017/2/23/T--Bielefeld-CeBiTec--protocol_Restriction_Digest.pdf">Restriction Digest</a></li> | ||
+ | <ul> | ||
+ | <li> 5 µL NEB2.1 buffer</li> | ||
+ | <li> 2 µL DNA</li> | ||
+ | <li> 1 µL <i>Xba</i>I</li> | ||
+ | <li> 1 µL <i>Pst</i>I</li> | ||
+ | <li> filled up to final volume 50 µL with ddH20</li> | ||
+ | <li> Incubation: 60 min, 37 °C</li> | ||
+ | <li> Heatinactivation: 20 min, 80 °C</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Aim of the Experiment </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Yannic Kerkhoff</p> | ||
+ | <p><b>Superior experiment:</b> GoTaq PCR of 32 clones of BBa_K2201200</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <li> Screening for positive transformations containing the Biobrick</li> | ||
+ | <ul> | ||
+ | <li> standard GoTaq-protocol</li> | ||
+ | <ul> | ||
+ | <li> template: colonie pcr clones of GA_K2201200</li> | ||
+ | <li> primer fwd: 17ns</li> | ||
+ | <li> primer rev: 17nt</li> | ||
+ | <li> annealing temperature: 56°C</li> | ||
+ | <li> extension time: 40 seconds</li> | ||
+ | </ul> | ||
+ | <li> positive clone 20</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Native PAGE of annealed primers (17jl & 17jm) </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Camilla Maerz</p> | ||
+ | <p><b>Superior experiment:</b> M.A.X restriction system - Primer annealing test</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> <a target="blank" href="https://static.igem.org/mediawiki/2017/e/e6/T--Bielefeld-CeBiTec--protocol_native_PAGE.pdf">Native DNA PAGE</a> standard protocol</li> | ||
+ | <ul> | ||
+ | <li> tested samples:</li> | ||
+ | <ul> | ||
+ | <li> HEPES annealing (17jl & 17jm): 100 µM, 10 µM, 5 µM, 2.5 µM, 1 µM, 0.5 µM</li> | ||
+ | <li> Aqua annealing (17jl & 17jm): 100 µM, 10 µM, 5 µM, 2.5 µM, 1 µM, 0.5 µM</li> | ||
+ | </ul> | ||
+ | <li> Variation: 60 V, 2 h</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> multiplication of plasmids containing the Biobrick </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Yannic Kerkhoff</p> | ||
+ | <p><b>Superior experiment:</b> preculture of BL21(DE3) BBa_K2201321</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> standard procedure</li> | ||
+ | <ul> | ||
+ | <li> 1,25µl Cam in 5mL LB</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> getting plasmids containing the Biobrick for further experiments </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Yannic Kerkhoff</p> | ||
+ | <p><b>Superior experiment:</b> plasmid isolation of preculture of BBa_K2201321 clone 3</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> standard protocol</li> | ||
+ | <ul> | ||
+ | <li> 319,1 ng/µL</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Native Page of annealed primers (17jl & 17jm; 17jh & 17ji) </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Camilla Maerz</p> | ||
+ | <p><b>Superior experiment:</b> M.A.X restriction system - Primer annealing test</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> <a target="blank" href="https://static.igem.org/mediawiki/2017/e/e6/T--Bielefeld-CeBiTec--protocol_native_PAGE.pdf">Native DNA PAGE</a> standard protocol</li> | ||
+ | <ul> | ||
+ | <li> tested samples:</li> | ||
+ | <ul> | ||
+ | <li> HEPES annealing (17jl & 17jm): 0.5 µM</li> | ||
+ | <li> Aqua annealing (17jl & 17jm): 0.5 µM</li> | ||
+ | <li> Composite Buffer annealing (17jh & 17 jm): 0.5 µM</li> | ||
+ | <li> all Primers (ssDNA): 1 µM</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-date"> | ||
+ | <h3> 2017-08-28 - 2017-09-03 </h3> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Native PAGE of annealed primers (HEPES annealing & Composite Buffer annealing) of the same concentration to test if all annealings have the same quality </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Camilla Maerz</p> | ||
+ | <p><b>Superior experiment:</b> M.A.X restriction system - Primer annealing test</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> all samples were diluted to a final concentration of 0.5 µM</li> | ||
+ | <li> <a target="blank" href="https://static.igem.org/mediawiki/2017/e/e6/T--Bielefeld-CeBiTec--protocol_native_PAGE.pdf">Native DNA PAGE</a> standard protocol</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> getting positive clones for further work </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Yannic Kerkhoff</p> | ||
+ | <p><b>Superior experiment:</b> heatshock transformation of the Biobrick Assembly product CP3</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> standard heatshock protocol</li> | ||
+ | <ul> | ||
+ | <li> used own produced chemo competend BL21(DE3)</li> | ||
+ | <li> streaked out on LB-plates with Cam</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Restriction digest of control annealings (mutC) </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Camilla Maerz</p> | ||
+ | <p><b>Superior experiment:</b> M.A.X restriction system</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> <a target="blank" href="https://static.igem.org/mediawiki/2017/2/23/T--Bielefeld-CeBiTec--protocol_Restriction_Digest.pdf">Restriction Digest</a> (<i>Mnl</i>I) of aqua annealing sample: mutC (0.5 µM)</li> | ||
+ | <ul> | ||
+ | <li> 2 µL <i>Mnl</i>I</li> | ||
+ | <li> 5 µL CutSmart</li> | ||
+ | <li> 25 µL mutC dsDNA</li> | ||
+ | <li> 18 µL nuclease-free H2O</li> | ||
+ | <li> Incubation: 37 °C, 1 h</li> | ||
+ | <li> Inactivation: 20 min., 65 °C</li> | ||
+ | </ul> | ||
+ | <li> <a target="blank" href="https://static.igem.org/mediawiki/2017/e/e6/T--Bielefeld-CeBiTec--protocol_native_PAGE.pdf">Native DNA PAGE</a> standard protocol</li> | ||
+ | <ul> | ||
+ | <li> Tested samples:</li> | ||
+ | <ul> | ||
+ | <li> digested mutC sample</li> | ||
+ | <li> native mutC sample</li> | ||
+ | <li> ssDNA primers (17jl & 17jm)</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> GoTaq PCR of 14 clones of BBa_K2201322 </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Yannic Kerkhoff</p> | ||
+ | <p><b>Superior experiment:</b> Screening for positive transformations containing the Biobrick</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> standard GoTaq-protocol</li> | ||
+ | <ul> | ||
+ | <li> template: colonie pcr clones of BBA_K2201322</li> | ||
+ | <li> primer fwd: Präfix_fwd</li> | ||
+ | <li> primer rev: Suffix_rev</li> | ||
+ | <li> annealing temperature: 56°C</li> | ||
+ | <li> extension time: 40 seconds</li> | ||
+ | </ul> | ||
+ | <li> positive: clone 3, 4, 5</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Restriction digest of aqua & HEPES annealing </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Camilla Maerz</p> | ||
+ | <p><b>Superior experiment:</b> M.A.X restriction system</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> <a target="blank" href="https://static.igem.org/mediawiki/2017/2/23/T--Bielefeld-CeBiTec--protocol_Restriction_Digest.pdf">Restriction Digest</a> (<i>Mnl</i>I) of aqua & HEPES annealing sample: mutC (0.5 µM)</li> | ||
+ | <ul> | ||
+ | <li> 2 µL <i>Mnl</i>I</li> | ||
+ | <li> 5 µL CutSmart</li> | ||
+ | <li> 25 µL mutC dsDNA</li> | ||
+ | <li> 18 µL nuclease-free H2O</li> | ||
+ | <li> Incubation: 37 °C, 1 h</li> | ||
+ | <li> Inactivation: 20 min., 65 °C</li> | ||
+ | </ul> | ||
+ | <li> <a target="blank" href="https://static.igem.org/mediawiki/2017/e/e6/T--Bielefeld-CeBiTec--protocol_native_PAGE.pdf">Native DNA PAGE</a> standard protocol</li> | ||
+ | <ul> | ||
+ | <li> Tested samples:</li> | ||
+ | <ul> | ||
+ | <li> digested mutC sample (aqua & HEPES annealing)</li> | ||
+ | <li> native mutC sample</li> | ||
+ | <li> ssDNA primers (17jl & 17jm)</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Aqua annealing of mutA, mutT, mutG and mutC </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Camilla Maerz</p> | ||
+ | <p><b>Superior experiment:</b> M.A.X restriction system</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> Aqua annealing standard protocol (final concentration: 0.5 µM)</li> | ||
+ | <ul> | ||
+ | <li> mutA: 17jf & 17jg</li> | ||
+ | <li> mutT: 17jj & 17jk</li> | ||
+ | <li> mutG: 17jh & 17ji</li> | ||
+ | <li> mutC: 17jl & 17jm</li> | ||
+ | </ul> | ||
+ | <li> final volume of each sample: 50 µL</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-date"> | ||
+ | <h3> 2017-09-04 - 2017-09-10 </h3> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Restriction digest of control annealings (mutA, mutT, mutG, mutC) and aqua annealing of oligo2 </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Camilla Maerz</p> | ||
+ | <p><b>Superior experiment:</b> M.A.X restriction system</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> Oligo2 annealing</li> | ||
+ | <ul> | ||
+ | <li> Aqua annealing standard protocol (final concentration: 0.5 µM)</li> | ||
+ | </ul> | ||
+ | <li> Restricted samples:</li> | ||
+ | <ul> | ||
+ | <li> <i>Mnl</i>I -> mutC, oligo2</li> | ||
+ | <li> <i>Sap</i>I -> mutG, oligo2</li> | ||
+ | <li> <i>Bsa</i>I -> mutT, oligo2</li> | ||
+ | <li> <i>Eci</i>I -> mutA, oligo2</li> | ||
+ | </ul> | ||
+ | <li> <a target="blank" href="https://static.igem.org/mediawiki/2017/2/23/T--Bielefeld-CeBiTec--protocol_Restriction_Digest.pdf">Restriction Digest</a>:</li> | ||
+ | <ul> | ||
+ | <li> 1 µL Enzyme</li> | ||
+ | <li> 5 µL CutSmart</li> | ||
+ | <li> 25 µL mutC dsDNA</li> | ||
+ | <li> 19 µL nuclease-free H2O</li> | ||
+ | <li> Incubation: 37 °C, 1 h</li> | ||
+ | <li> Inactivation: 20 min., 65 °C</li> | ||
+ | </ul> | ||
+ | <li> <a target="blank" href="https://static.igem.org/mediawiki/2017/e/e6/T--Bielefeld-CeBiTec--protocol_native_PAGE.pdf">Native DNA PAGE</a> standard protocol</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> SDS-Page and western blot of BBa_K2201321 </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Yannic Kerkhoff</p> | ||
+ | <p><b>Superior experiment:</b> SDS-Page and western blot of BBa_K2201321</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> standard SDS protocoll</li> | ||
+ | <li> standard Western blot protocoll</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> integration of the mRFP (BBa_J04450) in the aaRS plasmid as optical controle for the integration of an randomerized dsDNA </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Laura Schlueter</p> | ||
+ | <p><b>Superior experiment:</b> aaRS plasmid</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> amplification of the mRFP (Phusion Master Mix), Primer vg, vh, annealing temperature: 60°C</li> | ||
+ | <li> amplification of the library backbone(Phusion Master Mix), Primer 17ve, 17vf, Annealing temperature: 61°C</li> | ||
+ | <li> <a target="blank" href="https://static.igem.org/mediawiki/2017/5/55/T--Bielefeld-CeBiTec--protocol_gibson_assembly.pdf">Gibson Assembly</a> with Gibson Master Mix + mRFP (3.1 µL) + library backbone (1.9 µL)</li> | ||
+ | <li> transformation in DH5a via heatshock</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Generating the randomerized dsDNA for the library </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Laura Schlueter</p> | ||
+ | <p><b>Superior experiment:</b> aaRS plasmid</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> <a target="blank" href="https://static.igem.org/mediawiki/2017/d/d1/T--Bielefeld-CeBiTec--protocol_ssDNA_annealing.pdf">ssDNA Annealing</a> protocoll with the primers 17vi (1 µL), 17vj (1,4 µL)</li> | ||
+ | <li> agarose gelelectrophoresis, 3 %, positive result: bands of 120 bp</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Generating the library </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Laura Schlueter</p> | ||
+ | <p><b>Superior experiment:</b> aaRS plasmid</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> <a target="blank" href="https://static.igem.org/mediawiki/2017/5/55/T--Bielefeld-CeBiTec--protocol_gibson_assembly.pdf">Gibson Assembly</a> Master Mix + dsDNA (3.5 µl) + library Plasmid (0.5 µL) with the optical controle</li> | ||
+ | <li> transformation in DH5a via heatshock</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-date"> | ||
+ | <h3> 2017-09-11 - 2017-09-17 </h3> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Annealing, restriction digest and native PAGE of all samples </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Camilla Maerz</p> | ||
+ | <p><b>Superior experiment:</b> M.A.X restriction system</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> Aqua annealing standard protocol (final concentration: 0.5 µM)</li> | ||
+ | <ul> | ||
+ | <li> mutA: 17jf & 17jg</li> | ||
+ | <li> mutT: 17jj & 17jk</li> | ||
+ | <li> mutG: 17jh & 17ji</li> | ||
+ | <li> mutC: 17jl & 17jm</li> | ||
+ | <li> oligo2: olgio2_sense & oligo2_antisense</li> | ||
+ | </ul> | ||
+ | <li> final volume of each sample: 50 µL</li> | ||
+ | <li> Restricted samples:</li> | ||
+ | <ul> | ||
+ | <li> <i>Mnl</i>I -> mutC, oligo2</li> | ||
+ | <li> <i>Sap</i>I -> mutG, oligo2</li> | ||
+ | <li> <i>Bsa</i>I -> mutT, oligo2</li> | ||
+ | <li> <i>Eci</i>I -> mutA, oligo2</li> | ||
+ | </ul> | ||
+ | <li> <a target="blank" href="https://static.igem.org/mediawiki/2017/2/23/T--Bielefeld-CeBiTec--protocol_Restriction_Digest.pdf">Restriction Digest</a>:</li> | ||
+ | <ul> | ||
+ | <li> 1 µL Enzyme</li> | ||
+ | <li> 5 µL CutSmart</li> | ||
+ | <li> 25 µL mutC dsDNA</li> | ||
+ | <li> 19 µL nuclease-free H2O</li> | ||
+ | <li> Incubation: 37 °C, 1 h</li> | ||
+ | <li> Inactivation: 20 min., 65 °C</li> | ||
+ | </ul> | ||
+ | <li> <a target="blank" href="https://static.igem.org/mediawiki/2017/e/e6/T--Bielefeld-CeBiTec--protocol_native_PAGE.pdf">Native DNA PAGE</a> standard protocol</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Generating the library </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Laura Schlueter</p> | ||
+ | <p><b>Superior experiment:</b> aaRS plasmid</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> <a target="blank" href="https://static.igem.org/mediawiki/2017/5/55/T--Bielefeld-CeBiTec--protocol_gibson_assembly.pdf">Gibson Assembly</a> Master Mix + dsDNA (3.5 µl) + library Plasmid (0.5 µL) with the optical controle (8 x)</li> | ||
+ | <li> transformation in DH5a via heatshock (8 x)</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Generating the library </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Laura Schlueter</p> | ||
+ | <p><b>Superior experiment:</b> aaRS plasmid</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> <a target="blank" href="https://static.igem.org/mediawiki/2017/5/55/T--Bielefeld-CeBiTec--protocol_gibson_assembly.pdf">Gibson Assembly</a> Master Mix + dsDNA (3.5 µl) + library Plasmid (0.5 µL) with the opotical controle (16 x)</li> | ||
+ | <li> transformation in DH5a via heatshock (16 x)</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Generating the library </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Laura Schlueter</p> | ||
+ | <p><b>Superior experiment:</b> aaRS plasmid</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> <a target="blank" href="https://static.igem.org/mediawiki/2017/5/55/T--Bielefeld-CeBiTec--protocol_gibson_assembly.pdf">Gibson Assembly</a> Master Mix + dsDNA (3.5 µl) + library Plasmid (0.5 µL) with the opotical controle (25x)</li> | ||
+ | <li> transformation in DH5a via heatshock (25 x)</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Generating the library </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Laura Schlueter</p> | ||
+ | <p><b>Superior experiment:</b> aaRS plasmid</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> <a target="blank" href="https://static.igem.org/mediawiki/2017/5/55/T--Bielefeld-CeBiTec--protocol_gibson_assembly.pdf">Gibson Assembly</a> Master Mix + dsDNA (3.5 µl) + library Plasmid (0.5 µL) with the opotical controle (25x)</li> | ||
+ | <li> transformation in DH5a via heatshock (25 x)</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Generating the library </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Laura Schlueter</p> | ||
+ | <p><b>Superior experiment:</b> aaRS plasmid</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> <a target="blank" href="https://static.igem.org/mediawiki/2017/5/55/T--Bielefeld-CeBiTec--protocol_gibson_assembly.pdf">Gibson Assembly</a> Master Mix + dsDNA (3.5 µl) + library Plasmid (0.5 µL) with the opotical controle (25x)</li> | ||
+ | <li> transformation in DH5a via heatshock (25 x)</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Annealing, restriction digest and native PAGE of all samples </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Camilla Maerz</p> | ||
+ | <p><b>Superior experiment:</b> M.A.X restriction system</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> Aqua annealing standard protocol (final concentration: 0.5 µM)</li> | ||
+ | <ul> | ||
+ | <li> mutA: 17jf & 17jg</li> | ||
+ | <li> mutT: 17jj & 17jk</li> | ||
+ | <li> mutG: 17jh & 17ji</li> | ||
+ | <li> mutC: 17jl & 17jm</li> | ||
+ | <li> oligo2: olgio2_sense & oligo2_antisense</li> | ||
+ | </ul> | ||
+ | <li> final volume of each sample: 2x50 µL</li> | ||
+ | <li> Restricted samples:</li> | ||
+ | <ul> | ||
+ | <li> <i>Mnl</i>I -> mutC</li> | ||
+ | <li> <i>Sap</i>I -> mutG, oligo2</li> | ||
+ | <li> <i>Bsa</i>I -> mutT</li> | ||
+ | <li> <i>Eci</i>I -> mutA</li> | ||
+ | </ul> | ||
+ | <li> <a target="blank" href="https://static.igem.org/mediawiki/2017/2/23/T--Bielefeld-CeBiTec--protocol_Restriction_Digest.pdf">Restriction Digest</a>:</li> | ||
+ | <ul> | ||
+ | <li> 1 µL Enzyme</li> | ||
+ | <li> 5 µL CutSmart</li> | ||
+ | <li> 25 µL mutC dsDNA</li> | ||
+ | <li> 19 µL nuclease-free H2O</li> | ||
+ | <li> Incubation: 37 °C, 1 h</li> | ||
+ | <li> Inactivation: 20 min., 65 °C</li> | ||
+ | </ul> | ||
+ | <li> <a target="blank" href="https://static.igem.org/mediawiki/2017/e/e6/T--Bielefeld-CeBiTec--protocol_native_PAGE.pdf">Native DNA PAGE</a> standard protocol</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-date"> | ||
+ | <h3> 2017-09-18 - 2017-09-24 </h3> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> getting positive clones for further work </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Yannic Kerkhoff</p> | ||
+ | <p><b>Superior experiment:</b> heatshock transformation of the EutS-part from CU Boulder</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> standard heatshock protocol</li> | ||
+ | <ul> | ||
+ | <li> used own produced chemo competend DH5alpha</li> | ||
+ | <li> streaked out on LB-plates with Amp</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> getting positive clones for further work </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Yannic Kerkhoff</p> | ||
+ | <p><b>Superior experiment:</b> heatshock transformation of the FusionRedTag-part from CU Boulder</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> standard heatshock protocol</li> | ||
+ | <ul> | ||
+ | <li> used own produced chemo competend DH5alpha</li> | ||
+ | <li> streaked out on LB-plates with Kan</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> getting positive clones for further work </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Yannic Kerkhoff</p> | ||
+ | <p><b>Superior experiment:</b> heatshock cotransformation of the EutS-part and FusionRedTag-part from CU Boulder</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> standard heatshock protocol</li> | ||
+ | <ul> | ||
+ | <li> used own produced chemo competend DH5alpha</li> | ||
+ | <li> streaked out on LB-plates with AmpKan</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Agarose gel electrophoresis of digested mutA, mutT, mutG & mutC </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Camilla Maerz</p> | ||
+ | <p><b>Superior experiment:</b> M.A.X restriction system</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> Agarose gel electrophoresis (2%)</li> | ||
+ | <ul> | ||
+ | <li> 60 V, 40 min</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Annealing, restriction digest and native PAGE of all samples </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Camilla Maerz</p> | ||
+ | <p><b>Superior experiment:</b> M.A.X restriction system</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> Aqua annealing standard protocol (final concentration: 0.5 µM)</li> | ||
+ | <ul> | ||
+ | <li> mutA: 17jf & 17jg</li> | ||
+ | <li> mutT: 17jj & 17jk</li> | ||
+ | <li> mutG: 17jh & 17ji</li> | ||
+ | <li> mutC: 17jl & 17jm</li> | ||
+ | <li> oligo2: olgio2_sense & oligo2_antisense</li> | ||
+ | </ul> | ||
+ | <li> final volume of each sample: 2x50 µL</li> | ||
+ | <li> Restricted samples:</li> | ||
+ | <ul> | ||
+ | <li> <i>Mnl</i>I -> mutC</li> | ||
+ | <li> <i>Sap</i>I -> mutG, oligo2</li> | ||
+ | <li> <i>Bsa</i>I -> mutT</li> | ||
+ | <li> <i>Eci</i>I -> mutA</li> | ||
+ | </ul> | ||
+ | <li> <a target="blank" href="https://static.igem.org/mediawiki/2017/2/23/T--Bielefeld-CeBiTec--protocol_Restriction_Digest.pdf">Restriction Digest</a>:</li> | ||
+ | <ul> | ||
+ | <li> 1 µL Enzyme</li> | ||
+ | <li> 5 µL CutSmart</li> | ||
+ | <li> 25 µL mutC dsDNA</li> | ||
+ | <li> 19 µL nuclease-free H2O</li> | ||
+ | <li> Incubation: 37 °C, 1 h</li> | ||
+ | <li> Inactivation: 20 min., 65 °C</li> | ||
+ | </ul> | ||
+ | <li> <a target="blank" href="https://static.igem.org/mediawiki/2017/e/e6/T--Bielefeld-CeBiTec--protocol_native_PAGE.pdf">Native DNA PAGE</a> standard protocol</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Combination of two biobricks to get the part BBa_K2201200 in a tetracycline low copy plasmid </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Yannic Kerkhoff</p> | ||
+ | <p><b>Superior experiment:</b> Biobrick Assembly of BBa_K2201200 in psB3T5</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> <a target="blank" href="https://static.igem.org/mediawiki/2017/8/84/T--Bielefeld-CeBiTec--protocol_Standard_Biobrick.pdf">Standard BioBrick Assembly</a> protocol</li> | ||
+ | <ul> | ||
+ | <li> Backbone: pSB3T5, 2,0µL</li> | ||
+ | <li> upstream: BBa_K2201200, 3,6µL</li> | ||
+ | <li> Ligation time was 12 h at 8°C</li> | ||
+ | <ul> | ||
+ | <li> </li> | ||
+ | </ul> | ||
+ | <li> cultivation</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> glycerine stocks of K2201200, K2201220, K2201221, K2201320, K2201321 </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Yannic Kerkhoff</p> | ||
+ | <p><b>Superior experiment:</b> glycerine stocks of K2201200, K2201220, K2201221, K2201320, K2201321</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <li> glycerine stock standard protocol</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Screening for positive transformations containing the Biobrick </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Yannic Kerkhoff</p> | ||
+ | <p><b>Superior experiment:</b> GoTaq PCR of 30 clones of K2201200 in pSB3T5</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> standard GoTaq-protocol</li> | ||
+ | <ul> | ||
+ | <li> template: colonie pcr clones of K2201200 in pSB3T5</li> | ||
+ | <li> primer fwd: Präfix_fwd</li> | ||
+ | <li> primer rev: Suffix_rev</li> | ||
+ | <li> annealing temperature: 56°C</li> | ||
+ | <li> extension time: 40 seconds</li> | ||
+ | </ul> | ||
+ | <li> positive clones 1, 3, 6</li> | ||
+ | <ul> | ||
+ | <li> </li> | ||
+ | </ul> | ||
+ | <li> Preculture</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Colony PCR of pSB3K5_mRFP_link_ccdB and pSB1C3_PtNTT2(31-575)-GFP </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Camilla Maerz</p> | ||
+ | <p><b>Superior experiment:</b> Construction of pSB1C3-PlacUV5_PtNTT2</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> PCR</li> | ||
+ | <ul> | ||
+ | <li> Go Tag (Promega) protocol</li> | ||
+ | <li> Primer: VR, VF2</li> | ||
+ | <li> Danaturation: 56 °C</li> | ||
+ | <li> Elongation: 90 s & 180 s</li> | ||
+ | </ul> | ||
+ | <li> Agarose gel electrophoresis (1%)</li> | ||
+ | <ul> | ||
+ | <li> 100 V, 30 min</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-date"> | ||
+ | <h3> 2017-09-25 - 2017-10-01 </h3> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> getting positive clones for further work </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Yannic Kerkhoff</p> | ||
+ | <p><b>Superior experiment:</b> heatshock cotransformation of K2201200 in pSB3T5 and K2201321</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> standard heatshock protocol</li> | ||
+ | <ul> | ||
+ | <li> used own produced chemo competend BL21(DE3)</li> | ||
+ | <li> streaked out on LB-plates with TetCam</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> getting plasmids containing the Biobrick for further experiments </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Yannic Kerkhoff</p> | ||
+ | <p><b>Superior experiment:</b> plasmid isolation of preculture of BBa_K2201207</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> standard protocol</li> | ||
+ | <ul> | ||
+ | <li> 75,6 ng/µL</li> | ||
+ | <ul> | ||
+ | <li> </li> | ||
+ | </ul> | ||
+ | <li> Biobrick Assembly</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Aqua annealing of mutA-1, mutG-1, mutC-1, mutG-1 & oligo1 </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Camilla Maerz</p> | ||
+ | <p><b>Superior experiment:</b> M.A.X restriction system</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> Aqua annealing standard protocol (final concentration: 0.5 µM)</li> | ||
+ | <ul> | ||
+ | <li> mutA-1: 17iv & 17iw</li> | ||
+ | <li> mutT-1: 17jb & 17jc</li> | ||
+ | <li> mutG-1: 17ix & 17iy</li> | ||
+ | <li> mutC-1: 17jd & 17jc</li> | ||
+ | <li> oligo1: olgio1_sense & oligo1_antisense</li> | ||
+ | </ul> | ||
+ | <li> final volume: 50 µL</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> getting positive clones for further work </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Yannic Kerkhoff</p> | ||
+ | <p><b>Superior experiment:</b> heatshock cotransformation of K2201240 and K2201207</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> standard heatshock protocol</li> | ||
+ | <ul> | ||
+ | <li> used own produced chemo competend BL21(DE3)</li> | ||
+ | <li> streaked out on LB-plates with AmpCam</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> getting positive clones for further work </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Yannic Kerkhoff</p> | ||
+ | <p><b>Superior experiment:</b> heatshock cotransformation of K2201241 and K2201207</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> standard heatshock protocol</li> | ||
+ | <ul> | ||
+ | <li> used own produced chemo competend BL21(DE3)</li> | ||
+ | <li> streaked out on LB-plates with AmpCam</li> | ||
+ | <ul> | ||
+ | <li> </li> | ||
+ | </ul> | ||
+ | <li> Q5-PRC + Gel + cleanUp</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-date"> | ||
+ | <h3> 2017-10-02 - 2017-10-08 </h3> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> UBP-PCR with TiTaq </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Camilla Maerz</p> | ||
+ | <p><b>Superior experiment:</b> PCR with UBP</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> PCR</li> | ||
+ | <ul> | ||
+ | <li> PCR-UBP standard protocol (TiTaq)</li> | ||
+ | <li> samples:</li> | ||
+ | <ul> | ||
+ | <li> Ligation of: mutA, mutT, mutG, mutC and oligo 2 (1 ng/µL)</li> | ||
+ | </ul> | ||
+ | <li> Primer: 17vt & 17vu</li> | ||
+ | <li> Primer annealing: 56 °C</li> | ||
+ | <li> Elongation: 20 s</li> | ||
+ | </ul> | ||
+ | <li> PCR and DNA purification kit (Macherey-Nagel)</li> | ||
+ | <ul> | ||
+ | <li> Nucleo spin PCR and <a target="blank" href="https://static.igem.org/mediawiki/2017/0/0e/T--Bielefeld-CeBiTec--protocol_Monarch_PCR_and_DNA_clean_up_kit_NEB.pdf">Monarch® PCR & DNA Clean-up</a> protocol</li> | ||
+ | </ul> | ||
+ | <li> <a target="blank" href="https://static.igem.org/mediawiki/2017/2/23/T--Bielefeld-CeBiTec--protocol_Restriction_Digest.pdf">Restriction Digest</a></li> | ||
+ | <ul> | ||
+ | <li> 1 µL Enzyme</li> | ||
+ | <li> 5 µL CutSmart</li> | ||
+ | <li> 25 µL mutC dsDNA</li> | ||
+ | <li> 19 µL nuclease-free H2O</li> | ||
+ | <li> Incubation: 37 °C, 1 h</li> | ||
+ | <li> Inactivation: 20 min., 65 °C</li> | ||
+ | </ul> | ||
+ | <li> Restricted samples:</li> | ||
+ | <ul> | ||
+ | <li> <i>Mnl</i>I -> mutC</li> | ||
+ | <li> <i>Sap</i>I -> mutG</li> | ||
+ | <li> <i>Bsa</i>I -> mutT</li> | ||
+ | <li> <i>Eci</i>I -> mutA</li> | ||
+ | <li> <i>Eci</i>I, <i>Bsa</i>I, <i>Sap</i>I, <i>Mnl</i>I -> oligo2</li> | ||
+ | <li> Variation: oligo2 was digested with 0.5 µL of each enzyme</li> | ||
+ | </ul> | ||
+ | <li> Agarose gel electrophoresis (2 %)</li> | ||
+ | <ul> | ||
+ | <li> 100 V, 20 min</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Generating the library </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Laura Schlueter</p> | ||
+ | <p><b>Superior experiment:</b> aaRS plasmid</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> <a target="blank" href="https://static.igem.org/mediawiki/2017/5/55/T--Bielefeld-CeBiTec--protocol_gibson_assembly.pdf">Gibson Assembly</a> Master Mix + dsDNA (3.5 µl) + library Plasmid (0.5 µL) with the opotical controle (25x)</li> | ||
+ | <li> transformation in DH5a via heatshock (25 x)</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Kanymycin with amber stop codon </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Laura Schlueter</p> | ||
+ | <p><b>Superior experiment:</b> positiv selection plasmid</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> amplification (~850 bp) of the kanamycin with primers 17jo, 17jp (no second MET codon) 71°C</li> | ||
+ | <li> amplification (~850 bp) of the kanamycin with primers 17jr, 17jp (two amber stop codon) 71°C</li> | ||
+ | </ul> | ||
+ | <li> amplification of the K608007 backbone with the primers 17hb, 17hd 57°C</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Generating the library </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Laura Schlueter</p> | ||
+ | <p><b>Superior experiment:</b> aaRS plasmid</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> <a target="blank" href="https://static.igem.org/mediawiki/2017/5/55/T--Bielefeld-CeBiTec--protocol_gibson_assembly.pdf">Gibson Assembly</a> Master Mix + dsDNA (3.5 µl) + library Plasmid (0.5 µL) with the opotical controle (25x)</li> | ||
+ | <li> transformation in DH5a via heatshock (25 x)</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> UBP-PCR with GoTaq G2 and TiTaq </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Camilla Maerz</p> | ||
+ | <p><b>Superior experiment:</b> PCR with UBP</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> PCR with TiTaq:</li> | ||
+ | <ul> | ||
+ | <li> PCR-UBP standard protocol (TiTaq)</li> | ||
+ | <li> samples:</li> | ||
+ | <ul> | ||
+ | <li> Ligation of: mutA, mutT, mutG, mutC and oligo 2 (5 ng/µL)</li> | ||
+ | </ul> | ||
+ | <li> Primer: 17 vt & 17 vu</li> | ||
+ | <li> Primer annealing: 56 °C</li> | ||
+ | <li> Elongation: 20 s</li> | ||
+ | </ul> | ||
+ | <li> PCR with TiTaq:</li> | ||
+ | <ul> | ||
+ | <li> PCR-UBP standard protocol (TiTaq)</li> | ||
+ | <li> samples:</li> | ||
+ | <ul> | ||
+ | <li> Ligation of: mutA, mutT, mutG and mutC (5 ng/µL)</li> | ||
+ | </ul> | ||
+ | <li> Primer: VR & VF2</li> | ||
+ | <li> Primer annealing: 56 °C</li> | ||
+ | <li> Elongation: 20 s</li> | ||
+ | </ul> | ||
+ | <li> PCR with GoTaq:</li> | ||
+ | <ul> | ||
+ | <li> PCR-UBP standard protocol (GoTaq)</li> | ||
+ | <li> samples:</li> | ||
+ | <ul> | ||
+ | <li> Ligation of: mutA, mutT, mutG, mutC and oligo2 (5 ng/µL)</li> | ||
+ | </ul> | ||
+ | <li> Primer: 17 vt & 17 vu</li> | ||
+ | <li> Primer annealing: 56 °C</li> | ||
+ | <li> Elongation: 20 s</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> cloning of the aaRS in pSB3C5 (low copy) </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Laura Schlueter</p> | ||
+ | <p><b>Superior experiment:</b> aaRS growth experiment</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> restriction of pSB3C5 and aaRS+tRNA (NPA, AcF, Prk) with EcoRI and PstI following the restriction protocol</li> | ||
+ | <li> extraction from the gel (Macherey-Nagel Kit)</li> | ||
+ | <li> ligation of pSB3C5 and aaRS+tRNA (NPA, AcF, Prk) with T4 Ligase (NEB)</li> | ||
+ | <li> Trafo via heatshock</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> sceening of the kanamycin (03.10.2017) </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Laura Schlueter</p> | ||
+ | <p><b>Superior experiment:</b> positiv selection plasmid</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> Colony PCR (GoTaq, Primer VF2, VR, 56.5 °C)</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Restriction digest of ligated oligo2 </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Camilla Maerz</p> | ||
+ | <p><b>Superior experiment:</b> PCR with UBP</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> <a target="blank" href="https://static.igem.org/mediawiki/2017/2/23/T--Bielefeld-CeBiTec--protocol_Restriction_Digest.pdf">Restriction Digest</a></li> | ||
+ | <ul> | ||
+ | <li> 5 µL CutSmart</li> | ||
+ | <li> 30 µL ligated oligo2 DNA</li> | ||
+ | <li> 2 µL <i>Eco</i>RV</li> | ||
+ | <li> filled up to final volume 50 µL with ddH20</li> | ||
+ | <li> Incubation: 120 min, 37 °C</li> | ||
+ | <li> Inactivation: 20 min, 65 °C</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Gradient PCR of mutT </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Camilla Maerz</p> | ||
+ | <p><b>Superior experiment:</b> PCR with UBP</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> PCR</li> | ||
+ | <li> PCR-UBP standard protocol (GoTaq G2)</li> | ||
+ | <ul> | ||
+ | <li> sample: mutT (5 ng/µL)</li> | ||
+ | <li> Primer: 17 vt & 17 vu</li> | ||
+ | <li> Primer annealing: 50 - 56 °C</li> | ||
+ | <li> Elongation: 20 s</li> | ||
+ | </ul> | ||
+ | <li> Agarose gel electrophoresis (2%)</li> | ||
+ | <ul> | ||
+ | <li> 100 V, 30 min</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> PCR of mutA, mutT, mutG & mutC with UBP and oligo2 without UBP </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Camilla Maerz</p> | ||
+ | <p><b>Superior experiment:</b> PCR with UBP</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> PCR with TiTaq:</li> | ||
+ | <ul> | ||
+ | <li> PCR-UBP standard protocol (TiTaq)</li> | ||
+ | <li> samples:</li> | ||
+ | <ul> | ||
+ | <li> Ligation of: mutA, mutT, mutG and mutC (5 ng/µL), oligo2 (25 ng/µL)</li> | ||
+ | </ul> | ||
+ | <li> Primer: VR & VF2</li> | ||
+ | <li> Primer annealing: 56 °C</li> | ||
+ | <li> Elongation: 20 s</li> | ||
+ | <li> Variation:</li> | ||
+ | <ul> | ||
+ | <li> oligo2 without UBP, instead 1 µL H20 was added</li> | ||
+ | <li> 0.5 µL isoG & 0.5 µL isoCMe were added to mutA, mutT, mutG and mutC</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Generating the library </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Laura Schlueter</p> | ||
+ | <p><b>Superior experiment:</b> aaRS Plasmid</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> plasmid isolation of the Tyr-RS library-mix</li> | ||
+ | <ul> | ||
+ | <li> we stamped out the not randomized, negative, clones, easy to identify by the red colour because of the mRFP</li> | ||
+ | <li> washed from the cultivation plates with <a target="blank" href="https://static.igem.org/mediawiki/2017/a/a7/T--Bielefeld-CeBiTec--protocol_LB.pdf">LB-media</a></li> | ||
+ | </ul> | ||
+ | <li> restriction of the Library with EcoRI and PstI</li> | ||
+ | <li> proof of the restriction via agarose-gelelectrophoresis, 1 %</li> | ||
+ | <ul> | ||
+ | <li> positve result: bands on ~2000 bp (backbone) and ~1000 bp (Tyr-RS with NNK)</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Generating the library </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Laura Schlueter</p> | ||
+ | <p><b>Superior experiment:</b> aaRS plasmid</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> <a target="blank" href="https://static.igem.org/mediawiki/2017/5/55/T--Bielefeld-CeBiTec--protocol_gibson_assembly.pdf">Gibson Assembly</a> Master Mix + dsDNA (3.5 µl) + library Plasmid (0.5 µL) with the opotical controle (25x)</li> | ||
+ | <li> transformation in DH5a via heatshock (25 x)</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-date"> | ||
+ | <h3> 2017-10-09 - 2017-10-15 </h3> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> PCR with switched primers: VF2 & 17vu, VR & 17vt </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Camilla Maerz</p> | ||
+ | <p><b>Superior experiment:</b> PCR with UBP</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> PCR with TiTaq:</li> | ||
+ | <ul> | ||
+ | <li> PCR-UBP standard protocol (TiTaq)</li> | ||
+ | <li> samples:</li> | ||
+ | <ul> | ||
+ | <li> Ligation of: mutA, mutT, mutG and mutC (5 ng/µL), oligo2 (25 ng/µL)</li> | ||
+ | </ul> | ||
+ | <li> Primer: VF2 & 17vu, VR & 17vt</li> | ||
+ | <li> Primer annealing: 56 °C</li> | ||
+ | <li> Elongation: 20 s</li> | ||
+ | </ul> | ||
+ | <li> PCR with GoTaq:</li> | ||
+ | <ul> | ||
+ | <li> PCR-UBP standard protocol (GoTaq)</li> | ||
+ | <li> samples:</li> | ||
+ | <ul> | ||
+ | <li> Ligation of: mutA, mutT, mutG and mutC (5 ng/µL), oligo2 (25 ng/µL)</li> | ||
+ | </ul> | ||
+ | <li> Primer: VF2 & 17vu, VR & 17vt</li> | ||
+ | <li> Primer annealing: 56 °C</li> | ||
+ | <li> Elongation: 20 s</li> | ||
+ | </ul> | ||
+ | <li> Agarose gel electrophoresis (2%)</li> | ||
+ | <ul> | ||
+ | <li> 100 V, 30 min</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Generating the library </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Laura Schlueter</p> | ||
+ | <p><b>Superior experiment:</b> aaRS plasmid</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> <a target="blank" href="https://static.igem.org/mediawiki/2017/5/55/T--Bielefeld-CeBiTec--protocol_gibson_assembly.pdf">Gibson Assembly</a> Master Mix + dsDNA (3.5 µl) + library Plasmid (0.5 µL) with the opotical controle (25x)</li> | ||
+ | <li> transformation in DH5a via heatshock (25 x)</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> PCR with higher DNA template concentrations and gradient PCR of mutT </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Camilla Maerz</p> | ||
+ | <p><b>Superior experiment:</b> PCR with UBP</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> PCR with TiTaq:</li> | ||
+ | <ul> | ||
+ | <li> PCR-UBP standard protocol (TiTaq)</li> | ||
+ | <li> samples:</li> | ||
+ | <ul> | ||
+ | <li> Ligation of: mutT (5 ng/µL) and oligo2 (50 ng/µL)</li> | ||
+ | </ul> | ||
+ | <li> Primer: VF2 & 17vu</li> | ||
+ | <li> Primer annealing: 56 °C</li> | ||
+ | <li> Elongation: 20 s</li> | ||
+ | </ul> | ||
+ | <li> PCR and DNA purification kit (Macherey-Nagel)</li> | ||
+ | <ul> | ||
+ | <li> Nucleo spin PCR and <a target="blank" href="https://static.igem.org/mediawiki/2017/0/0e/T--Bielefeld-CeBiTec--protocol_Monarch_PCR_and_DNA_clean_up_kit_NEB.pdf">Monarch® PCR & DNA Clean-up</a> protocol</li> | ||
+ | </ul> | ||
+ | <li> <a target="blank" href="https://static.igem.org/mediawiki/2017/2/23/T--Bielefeld-CeBiTec--protocol_Restriction_Digest.pdf">Restriction Digest</a></li> | ||
+ | <ul> | ||
+ | <li> 0.5 µL Enzyme</li> | ||
+ | <li> 2.3 µL CutSmart</li> | ||
+ | <li> 20 µL dsDNA</li> | ||
+ | <li> Incubation: 37 °C, 1 h</li> | ||
+ | <li> Inactivation: 20 min., 65 °C</li> | ||
+ | </ul> | ||
+ | <li> Restricted samples:</li> | ||
+ | <ul> | ||
+ | <li> <i>Bsa</i>I -> mutT</li> | ||
+ | <li> <i>Eci</i>I, <i>Bsa</i>I, <i>Sap</i>I, <i>Mnl</i>I -> oligo2</li> | ||
+ | <li> Variation: oligo2 was digested with 0.5 µL of each enzyme</li> | ||
+ | </ul> | ||
+ | <li> Agarose gel electrophoresis (2 %)</li> | ||
+ | <ul> | ||
+ | <li> 100 V, 20 min</li> | ||
+ | </ul> | ||
+ | <li> Gradient PCR with TiTaq:</li> | ||
+ | <ul> | ||
+ | <li> PCR-UBP standard protocol (TiTaq)</li> | ||
+ | <li> samples:</li> | ||
+ | <ul> | ||
+ | <li> Ligation of: mutT (5 ng/µL)</li> | ||
+ | </ul> | ||
+ | <li> Primer: VF2 & 17vu</li> | ||
+ | <li> Primer annealing: 50 - 58 °C</li> | ||
+ | <li> Elongation: 20 s</li> | ||
+ | </ul> | ||
+ | <li> Agarose gel electrophoresis (2%)</li> | ||
+ | <ul> | ||
+ | <li> 100 V, 30 min</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> PCR of mutA, mutT, mutG, mutC and oligo2 </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Camilla Maerz</p> | ||
+ | <p><b>Superior experiment:</b> PCR with UBP</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> PCR with GoTaq:</li> | ||
+ | <ul> | ||
+ | <li> PCR-UBP standard protocol (GoTaq)</li> | ||
+ | <li> samples:</li> | ||
+ | <ul> | ||
+ | <li> Ligation of: mutA, mutT, mutG, mutC (5 ng/µL) and oligo2 (25 ng/µL)</li> | ||
+ | </ul> | ||
+ | <li> Primer: VF2 & 17vu</li> | ||
+ | <li> Primer annealing: 56 °C</li> | ||
+ | <li> Elongation: 20 s</li> | ||
+ | </ul> | ||
+ | <li> PCR and DNA purification kit (Macherey-Nagel)</li> | ||
+ | <ul> | ||
+ | <li> Nucleo spin PCR and <a target="blank" href="https://static.igem.org/mediawiki/2017/0/0e/T--Bielefeld-CeBiTec--protocol_Monarch_PCR_and_DNA_clean_up_kit_NEB.pdf">Monarch® PCR & DNA Clean-up</a> protocol</li> | ||
+ | </ul> | ||
+ | <li> <a target="blank" href="https://static.igem.org/mediawiki/2017/2/23/T--Bielefeld-CeBiTec--protocol_Restriction_Digest.pdf">Restriction Digest</a></li> | ||
+ | <ul> | ||
+ | <li> 1 µL Enzyme</li> | ||
+ | <li> 2.3 µL CutSmart</li> | ||
+ | <li> 20 µL dsDNA</li> | ||
+ | <li> Incubation: 37 °C, 12 h</li> | ||
+ | <li> Inactivation: 20 min., 65 °C</li> | ||
+ | </ul> | ||
+ | <li> Restricted samples:</li> | ||
+ | <ul> | ||
+ | <li> <i>Mnl</i>I -> mutC</li> | ||
+ | <li> <i>Sap</i>I -> mutG</li> | ||
+ | <li> <i>Bsa</i>I -> mutT</li> | ||
+ | <li> <i>Eci</i>I -> mutA</li> | ||
+ | <li> <i>Eci</i>I, <i>Bsa</i>I, <i>Sap</i>I, <i>Mnl</i>I -> oligo2</li> | ||
+ | </ul> | ||
+ | <li> Agarose gel electrophoresis (2 %)</li> | ||
+ | <ul> | ||
+ | <li> 100 V, 30 min</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> positive selection with the library </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Laura Schlueter</p> | ||
+ | <p><b>Superior experiment:</b> pos selection</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> using cultivation plates with kan (0.5 mM), Cm (0.25 mM), Tet (0.5 mM)</li> | ||
+ | <ul> | ||
+ | <li> no growth</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> Variation of restriction digest </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Camilla Maerz</p> | ||
+ | <p><b>Superior experiment:</b> PCR with UBP</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> <a target="blank" href="https://static.igem.org/mediawiki/2017/2/23/T--Bielefeld-CeBiTec--protocol_Restriction_Digest.pdf">Restriction Digest</a></li> | ||
+ | <ul> | ||
+ | <li> 3 µL Enzyme</li> | ||
+ | <li> 2.5 µL CutSmart</li> | ||
+ | <li> 20 µL dsDNA</li> | ||
+ | <li> Incubation: 37 °C, 1 h</li> | ||
+ | <li> Inactivation: 20 min., 65 °C</li> | ||
+ | </ul> | ||
+ | <li> Restricted samples:</li> | ||
+ | <ul> | ||
+ | <li> <i>Mnl</i>I -> mutC</li> | ||
+ | <li> <i>Sap</i>I -> mutG</li> | ||
+ | <li> <i>Bsa</i>I -> mutT</li> | ||
+ | <li> <i>Eci</i>I -> mutA</li> | ||
+ | <li> <i>Eci</i>I, <i>Bsa</i>I, <i>Sap</i>I, <i>Mnl</i>I -> oligo2</li> | ||
+ | </ul> | ||
+ | <li> Agarose gel electrophoresis (2 %)</li> | ||
+ | <ul> | ||
+ | <li> 100 V, 30 min</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> positive selection with the library </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Laura Schlueter</p> | ||
+ | <p><b>Superior experiment:</b> pos selection</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> Transformation (heatshock) of the library plasmid mix in competent cells containing the positive selction plasmid</li> | ||
+ | <li> using cultivation plates with kan (0.3 mM), Cm (0.25 mM), Tet (0.5 mM)</li> | ||
+ | <ul> | ||
+ | <li> no growth</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> positive selection with the library </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Laura Schlueter</p> | ||
+ | <p><b>Superior experiment:</b> pos selection</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> Transformation (heatshock) of the library plasmid mix in competent cells containing the positive selction plasmid</li> | ||
+ | <li> extended regeneration time: adding 0,5 mM IPTG after 1 h in <a target="blank" href="https://static.igem.org/mediawiki/2017/f/fe/T--Bielefeld-CeBiTec--protocol_SOC.pdf">SOC media</a></li> | ||
+ | <li> using cultivation plates with kan (0.3 mM), Cm (0.25 mM), Tet (0.5 mM), IPTG (0,5 mM)</li> | ||
+ | <ul> | ||
+ | <li> no growth</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> growth experiments with the aaRS </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Laura Schlueter</p> | ||
+ | <p><b>Superior experiment:</b> pos selection</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> comparing AcF-TAG, AcF-Leu, Cou-AS, Prk, NPA (on low and high copy plasmid) pSB1C3, pSB3T5</li> | ||
+ | <ul> | ||
+ | <li> cultivation:</li> | ||
+ | <ul> | ||
+ | <li> <a target="blank" href="https://static.igem.org/mediawiki/2017/a/a7/T--Bielefeld-CeBiTec--protocol_LB.pdf">LB media</a>, Cm (0.25 mM) / Tet (0.5 mM) and 1 mM of the ncAA</li> | ||
+ | <li> 12 microtiter well plate, 600 rpm, 37°C</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-date"> | ||
+ | <h3> 2017-10-16 - 2017-10-22 </h3> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> PCR with TiTaq and Q5-polymerase </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Camilla Maerz</p> | ||
+ | <p><b>Superior experiment:</b> PCR with UBP</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> PCR with TiTaq:</li> | ||
+ | <ul> | ||
+ | <li> PCR-UBP standard protocol (TiTaq)</li> | ||
+ | <li> samples:</li> | ||
+ | <ul> | ||
+ | <li> Ligation of: mutA, mutT, mutG & mutC (5 ng/µL) and oligo2 (50 ng/µL)</li> | ||
+ | </ul> | ||
+ | <li> Primer: VF2 & 17vu</li> | ||
+ | <li> Primer annealing: 56 °C</li> | ||
+ | <li> Elongation: 20 s</li> | ||
+ | </ul> | ||
+ | <li> PCR and DNA purification kit (Macherey-Nagel)</li> | ||
+ | <ul> | ||
+ | <li> Nucleo spin PCR and <a target="blank" href="https://static.igem.org/mediawiki/2017/0/0e/T--Bielefeld-CeBiTec--protocol_Monarch_PCR_and_DNA_clean_up_kit_NEB.pdf">Monarch® PCR & DNA Clean-up</a> protocol</li> | ||
+ | </ul> | ||
+ | <li> <a target="blank" href="https://static.igem.org/mediawiki/2017/8/88/T--Bielefeld-CeBiTec--protocol_PCR_Q5_HF_Polymerase.pdf">PCR with Q5 High-Fidelity Polymerase</a>-polymerase (per reaction, 25 µL):</li> | ||
+ | <ul> | ||
+ | <li> 5 µL Reaction buffer</li> | ||
+ | <li> 1.25 µL dNTPs (2 mM)</li> | ||
+ | <li> 1.25 µL 3'-Primer (10 mM)</li> | ||
+ | <li> 1.25 µL 5'-Primer (10 mM)</li> | ||
+ | <li> 1 µL DNA template</li> | ||
+ | <li> 0.25 µL Q5 Hifi-Polymerase</li> | ||
+ | <li> 14 µL H20</li> | ||
+ | <li> mutA, mutT, mutG, mutC: + 1 µL H20</li> | ||
+ | <li> oligo2: + 0.5 µL isoCme, + 0.5 µL isoG</li> | ||
+ | <li> Primer: VF2 & 17vu</li> | ||
+ | <li> Primer annealing: 56 °C</li> | ||
+ | <li> Elongation: 10 s</li> | ||
+ | </ul> | ||
+ | <li> PCR and DNA purification kit (Macherey-Nagel)</li> | ||
+ | <ul> | ||
+ | <li> Nucleo spin PCR and <a target="blank" href="https://static.igem.org/mediawiki/2017/0/0e/T--Bielefeld-CeBiTec--protocol_Monarch_PCR_and_DNA_clean_up_kit_NEB.pdf">Monarch® PCR & DNA Clean-up</a> protocol</li> | ||
+ | </ul> | ||
+ | <li> <a target="blank" href="https://static.igem.org/mediawiki/2017/2/23/T--Bielefeld-CeBiTec--protocol_Restriction_Digest.pdf">Restriction Digest</a></li> | ||
+ | <ul> | ||
+ | <li> 1 µL Enzyme</li> | ||
+ | <li> 20 µL dsDNA</li> | ||
+ | <li> 2.3 µL CutSmart Buffer</li> | ||
+ | <li> Incubation: 37 °C, 15 h (<i>Bsa</i>I & <i>Mnl</i>I), 2 h (<i>Sap</i>I & <i>Eci</i>I)</li> | ||
+ | <li> Inactivation: 65 °C, 20 min</li> | ||
+ | </ul> | ||
+ | <li> Restricted samples:</li> | ||
+ | <ul> | ||
+ | <li> <i>Mnl</i>I -> mutC</li> | ||
+ | <li> <i>Sap</i>I -> mutG</li> | ||
+ | <li> <i>Bsa</i>I -> mutT</li> | ||
+ | <li> <i>Eci</i>I -> mutA</li> | ||
+ | <li> <i>Eci</i>I, <i>Bsa</i>I, <i>Sap</i>I, <i>Mnl</i>I -> oligo2</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> PCR with different polymerases </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Camilla Maerz</p> | ||
+ | <p><b>Superior experiment:</b> PCR with UBP</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> samples:</li> | ||
+ | <ul> | ||
+ | <li> Ligation of: mutA, mutT, mutG & mutC (5 ng/µL) and oligo2 (50 ng/µL)</li> | ||
+ | </ul> | ||
+ | <li> PCR with Allin Hifi DNA Polymerase (per reaction, 25 µL):</li> | ||
+ | <ul> | ||
+ | <li> 5 µL ReactionBuffer</li> | ||
+ | <li> 0.25 µL Hifi DNA Polymerase</li> | ||
+ | <li> 2.5 µL 3'-Primer (10 mM)</li> | ||
+ | <li> 2.5 µL 5'-Primer (10 mM)</li> | ||
+ | <li> 1 µL DNA template</li> | ||
+ | <li> 12.75 µL H20</li> | ||
+ | <li> mutA, mutT, mutG, mutC: + 1 µL H20</li> | ||
+ | <li> oligo2: + 0.5 µL isoCme, + 0.5 µL isoG</li> | ||
+ | <li> Primer: VF2 & 17vu</li> | ||
+ | <li> Primer annealing: 56 °C</li> | ||
+ | <li> Elongation: 10 s</li> | ||
+ | </ul> | ||
+ | <li> PCR and DNA purification kit (Macherey-Nagel)</li> | ||
+ | <ul> | ||
+ | <li> Nucleo spin PCR and <a target="blank" href="https://static.igem.org/mediawiki/2017/0/0e/T--Bielefeld-CeBiTec--protocol_Monarch_PCR_and_DNA_clean_up_kit_NEB.pdf">Monarch® PCR & DNA Clean-up</a> protocol</li> | ||
+ | </ul> | ||
+ | <li> PCR with innuDRY Standard PCR MasterMix (per reaction, 20 µL):</li> | ||
+ | <ul> | ||
+ | <li> 10 µL innduDRY Mastermix</li> | ||
+ | <li> 2 µL 3'-Primer (10 mM)</li> | ||
+ | <li> 2 µL 5'-Primer (10 mM)</li> | ||
+ | <li> 1 µL DNA template</li> | ||
+ | <li> 5 µL H20</li> | ||
+ | <li> mutA, mutT, mutG, mutC: + 1 µL H20</li> | ||
+ | <li> oligo2: + 0.5 µL isoCme, + 0.5 µL isoG</li> | ||
+ | <li> Primer: VF2 & 17vu</li> | ||
+ | <li> Primer annealing: 54 °C</li> | ||
+ | <li> Elongation: 30 s</li> | ||
+ | </ul> | ||
+ | <li> PCR and DNA purification kit (Macherey-Nagel)</li> | ||
+ | <ul> | ||
+ | <li> Nucleo spin PCR and <a target="blank" href="https://static.igem.org/mediawiki/2017/0/0e/T--Bielefeld-CeBiTec--protocol_Monarch_PCR_and_DNA_clean_up_kit_NEB.pdf">Monarch® PCR & DNA Clean-up</a> protocol</li> | ||
+ | </ul> | ||
+ | <li> PCR with BioMaster-HS Taq PCR Color (per reaction, 25 µL):</li> | ||
+ | <ul> | ||
+ | <li> 12.5 µL Mastermix</li> | ||
+ | <li> 0.7 µL 3'-Primer (10 mM)</li> | ||
+ | <li> 0.7 µL 5'-Primer (10 mM)</li> | ||
+ | <li> 1 µL DNA template</li> | ||
+ | <li> 10.1 µL H20</li> | ||
+ | <li> mutA, mutT, mutG, mutC: + 1 µL H20</li> | ||
+ | <li> oligo2: + 0.5 µL isoCme, + 0.5 µL isoG</li> | ||
+ | <li> Primer: VF2 & 17vu</li> | ||
+ | <li> Primer annealing: 51 °C</li> | ||
+ | <li> Elongation: 20 s</li> | ||
+ | </ul> | ||
+ | <li> PCR and DNA purification kit (Macherey-Nagel)</li> | ||
+ | <ul> | ||
+ | <li> Nucleo spin PCR and <a target="blank" href="https://static.igem.org/mediawiki/2017/0/0e/T--Bielefeld-CeBiTec--protocol_Monarch_PCR_and_DNA_clean_up_kit_NEB.pdf">Monarch® PCR & DNA Clean-up</a> protocol</li> | ||
+ | </ul> | ||
+ | <li> PCR with Fire Polymerase (per reaction, 20 µL):</li> | ||
+ | <ul> | ||
+ | <li> 4 µL Mastermix</li> | ||
+ | <li> 0.5 µL 3'-Primer (10 mM)</li> | ||
+ | <li> 0.5 µL 5'-Primer (10 mM)</li> | ||
+ | <li> 1 µL DNA template</li> | ||
+ | <li> 14 µL H20</li> | ||
+ | <li> mutA, mutT, mutG, mutC: + 1 µL H20</li> | ||
+ | <li> oligo2: + 0.5 µL isoCme, + 0.5 µL isoG</li> | ||
+ | <li> Primer: VF2 & 17vu</li> | ||
+ | <li> Primer annealing: 56 °C</li> | ||
+ | <li> Elongation: 20 s</li> | ||
+ | </ul> | ||
+ | <li> PCR and DNA purification kit (Macherey-Nagel)</li> | ||
+ | <ul> | ||
+ | <li> Nucleo spin PCR and <a target="blank" href="https://static.igem.org/mediawiki/2017/0/0e/T--Bielefeld-CeBiTec--protocol_Monarch_PCR_and_DNA_clean_up_kit_NEB.pdf">Monarch® PCR & DNA Clean-up</a> protocol</li> | ||
+ | </ul> | ||
+ | <li> PCR with Phusion polymerase (per reaction, 25 µL):</li> | ||
+ | <ul> | ||
+ | <li> 12.5 µL Mastermix</li> | ||
+ | <li> 1.25 µL 3'-Primer (10 mM)</li> | ||
+ | <li> 1.25 µL 5'-Primer (10 mM)</li> | ||
+ | <li> 1 µL DNA template</li> | ||
+ | <li> 9 µL H20</li> | ||
+ | <li> mutA, mutT, mutG, mutC: + 1 µL H20</li> | ||
+ | <li> oligo2: + 0.5 µL isoCme, + 0.5 µL isoG</li> | ||
+ | <li> Primer: VF2 & 17vu</li> | ||
+ | <li> Primer annealing: 56 °C</li> | ||
+ | <li> Elongation: 20 s</li> | ||
+ | </ul> | ||
+ | <li> PCR and DNA purification kit (Macherey-Nagel)</li> | ||
+ | <ul> | ||
+ | <li> Nucleo spin PCR and <a target="blank" href="https://static.igem.org/mediawiki/2017/0/0e/T--Bielefeld-CeBiTec--protocol_Monarch_PCR_and_DNA_clean_up_kit_NEB.pdf">Monarch® PCR & DNA Clean-up</a> protocol</li> | ||
+ | </ul> | ||
+ | <li> <a target="blank" href="https://static.igem.org/mediawiki/2017/2/23/T--Bielefeld-CeBiTec--protocol_Restriction_Digest.pdf">Restriction Digest</a></li> | ||
+ | <ul> | ||
+ | <li> 1 µL Enzyme</li> | ||
+ | <li> 20 µL dsDNA</li> | ||
+ | <li> 2.3 µL CutSmart Buffer</li> | ||
+ | <li> Incubation: 37 °C, 15 h (<i>Bsa</i>I & <i>Mnl</i>I), 2 h (<i>Sap</i>I & <i>Eci</i>I)</li> | ||
+ | <li> Inactivation: 65 °C, 20 min</li> | ||
+ | </ul> | ||
+ | <li> Restricted samples:</li> | ||
+ | <ul> | ||
+ | <li> <i>Mnl</i>I -> mutC</li> | ||
+ | <li> <i>Sap</i>I -> mutG</li> | ||
+ | <li> <i>Bsa</i>I -> mutT</li> | ||
+ | <li> <i>Eci</i>I -> mutA</li> | ||
+ | <li> <i>Eci</i>I, <i>Bsa</i>I, <i>Sap</i>I, <i>Mnl</i>I -> oligo2</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="labnotebox"> | ||
+ | <div class="labnote-title"> | ||
+ | <h3> PCR with different polymerases </h3> | ||
+ | </div> | ||
+ | <div class="labnote-content"> | ||
+ | <p><b>Investigators:</b> Camilla Maerz</p> | ||
+ | <p><b>Superior experiment:</b> PCR with UBP</p> | ||
+ | <p><b>Procedure:</b></p> | ||
+ | <article> | ||
+ | <ul> | ||
+ | <li> Agarose gel electrophoresis with all samples (17.10)</li> | ||
+ | <li> Agarose gel electrophoresis (2%)</li> | ||
+ | <ul> | ||
+ | <li> 100 V, 40 min</li> | ||
+ | </ul> | ||
+ | </article> | ||
+ | </div> | ||
+ | </div> | ||
+ | |||
+ | |||
+ | |||
</div> | </div> | ||
</body> | </body> | ||
+ | <script> | ||
+ | $("#notebook").addClass("active"); | ||
+ | $("#notebook-labjournal").addClass("active"); | ||
+ | </script> | ||
</html> | </html> | ||
{{Team:Bielefeld-CeBiTec/Footer}} | {{Team:Bielefeld-CeBiTec/Footer}} |
Latest revision as of 03:56, 2 November 2017
2017-04-03 - 2017-04-09
getting positive clones for further work
Investigators: Yannic Kerkhoff
Superior experiment: electroporation transformation of BBa_K1416000
Procedure:
getting positive clones for further work
Investigators: Yannic Kerkhoff
Superior experiment: electroporation transformation of BBa_KJ36848
Procedure:
- standard electroporation protocol
- used electro competend DH5alpha from NEB
- streaked out on LB-plates with Cam
multiplication of plasmids containing the Biobrick
Investigators: Yannic Kerkhoff
Superior experiment: preculture of transformed BBa_K1416000
Procedure:
- standard procedure
- 1,25µl Cam in 5mL LB
2017-04-10 - 2017-04-16
getting plasmids containing the Biobrick for further experiments
Investigators: Yannic Kerkhoff
Superior experiment: plasmid isolation of preculture of BBa_K1416000
Procedure:
- standard protocol
- 517,9 ng/µL
multiplication of plasmids containing the Biobrick
Investigators: Yannic Kerkhoff
Superior experiment: preculture of transformed BBa_J36848
Procedure:
- standard procedure
- 1,25µl Cam in 5mL LB
getting plasmids containing the Biobrick for further experiments
Investigators: Yannic Kerkhoff
Superior experiment: plasmid isolation of preculture of BBa_J36848
Procedure:
- standard protocol
- 231,8 ng/µL
2017-05-29 - 2017-06-04
getting positive clones for further work
Investigators: Yannic Kerkhoff
Superior experiment: heatshock transformation of BBa_E0400
Procedure:
- standard heatshock protocol
- used own produced chemo competend DH5alpha
- streaked out on LB-plates with Amp
2017-06-05 - 2017-06-11
multiplication of plasmids containing the Biobrick
Investigators: Yannic Kerkhoff
Superior experiment: preculture of transformed BBa_E0400
Procedure:
- standard procedure
- 5,0µl Amp in 5mL LB
getting plasmids containing the Biobrick for further experiments
Investigators: Yannic Kerkhoff
Superior experiment: plasmid isolation of preculture of BBa_E0400
Procedure:
- standard protocol
- 88,4 ng/µL
getting positive clones for further work
Investigators: Yannic Kerkhoff
Superior experiment: heatshock transformation of BBa_K525998
Procedure:
- standard heatshock protocol
- used own produced chemo competend DH5alpha
- streaked out on LB-plates with Cam
multiplication of plasmids containing the Biobrick
Investigators: Yannic Kerkhoff
Superior experiment: preculture of transformed BBa_K525998
Procedure:
- standard procedure
- 1,25µl Cam in 5mL LB
getting plasmids containing the Biobrick for further experiments
Investigators: Yannic Kerkhoff
Superior experiment: plasmid isolation of preculture of BBa_K525998
Procedure:
- standard protocol
- 140,9 ng/µL
2017-06-12 - 2017-06-18
Assemble the fragments to get the Part BBa_K2201220
Investigators: Yannic Kerkhoff
Superior experiment: Gibson assembly of F1,F3,F5
Procedure:
- standard Gibson Assembly protocol
Cellgrowth with the barnaseplasmid
Investigators: Olga Schmidt, Denise Kerkhoff, Christina Drake
Superior experiment: Overnightculture of barnase
Procedure:
- 3 ml LB-medium and 0,75 µl chloramphenicol
- inkubation overnight 37°C, 140rpm
To get and purified the plasmid
Investigators: Christina Drake
Superior experiment: Plasmidisolation of barnase-plasmid
Procedure:
- protocol from Macherley-Nagel: Plasmid DNA purification, protocol 5: NucleoSpin
- concentration measurement by nanoprop
- sequenzingorder
Duplicate the parts
Investigators: Christina Drake, Denise Kerkhoff
Superior experiment: Transformation via Distribution of the parts BBA_J04450, BBa_I746909, BBa_K80800 and BBa_psB1K5
Procedure:
- transformation via electroporation
- plated out: BBa_J04450 on tetracycline, BBa_I74909 and BBa_K808000 on chloramphenicol, BBa_psB1K5 on kanamycin
Duplicate the part
Investigators: Christina Drake, Denise Kerkhoff
Superior experiment: Transformation of BBa_psB6A1
Procedure:
- transformation via electroporation
- plated out on amphenicol
Cellgrowth with the plasmids
Investigators: Denise Kerkhoff, Christina Drake
Superior experiment: Overnightculture of BBA_J04450, BBa_I746909 and BBa_K808000
Procedure:
- 3 ml LB-medium and at BBA_J04450 3µl tetracycline and atBBa_I746909 and BBa_K808000 0,75 µl chloramphenicol
- inkubation overnight 37°C, 140rpm
To get and purified the plasmid
Investigators: Christina Drake
Superior experiment: Plasmidisolation of BBA_J04450, BBa_I746909 and BBa_K80800-plasmids
Procedure:
- protocol from Macherley-Nagel: Plasmid DNA purification, protocol 5: NucleoSpin
- concentration measurement by nanoprop:
- BBA_J04450 26ng/µl and 25,7ng/µl
- BBa_I746909 90,2ng/µl
- 104,3 ng/µl -BBa_K808000 118,3 ng/µl
- and 146,6 ng/µl
- sequenzingorder
2017-06-19 - 2017-06-25
Cellgrowth with the plasmid
Investigators: Denise Kerkhoff
Superior experiment: Overnightculture of BBA_psB3K5
Procedure:
- 3 ml LB-medium and 2,5 µl kanamycin
- inkubation overnight 37°C, 140rpm
getting positive clones for further work
Investigators: Yannic Kerkhoff
Superior experiment: heatshock transformation of assembled fragments F5,F2,F4
Procedure:
- standard heatshock protocol
- used own produced chemo competend DH5alpha
- streaked out on LB-plates with Cam
multiplication, verification and purification of the needed fragment F2: GFPLinker2
Investigators: Yannic Kerkhoff
Superior experiment: Q5-PCR, gel and PCR-cleanUp of F2
Procedure:
- standard Q5-PCR protocol
- template: BBa_E0400
- primer fwd: 17gk
- primer rev: 17gn
- annealing temperature: 58°C
- extension time: 50 seconds
- standard PCR-cleanUp protocol
- 125,8 ng/µL
multiplication, verification and purification of the needed fragment F4: StrepLinker2
Investigators: Yannic Kerkhoff
Superior experiment: Q5-PCR, gel and PCR-cleanUp of F4
Procedure:
- standard Q5-PCR protocol
- template: BBa_KJ36848
- primer fwd: 17gp
- primer rev: 17gq
- annealing temperature: 61°C
- extension time: 30 seconds
- standard PCR-cleanUp protocol
- 127,0 ng/µL
multiplication, verification and purification of the needed fragment F5: linearized pSB1C3
Investigators: Yannic Kerkhoff
Superior experiment: Q5-PCR, gel and PCR-cleanUp of F5
Procedure:
- standard Q5-PCR protocol
- template: K1416000
- primer fwd: 17gj
- primer rev: 17gl
- annealing temperature: 59°C
- extension time: 120 seconds
- standard PCR-cleanUp protocol
- 258,4 ng/µL
Assemble the fragments to get the Part BBa_K2201221
Investigators: Yannic Kerkhoff
Superior experiment: Gibson assembly of F2,F4,F5
Procedure:
- standard Gibson Assembly protocol
Screening for positive transformations containing the Biobrick
Investigators: Yannic Kerkhoff
Superior experiment: GoTaq PCR of 7 clones of BBa_K2201221
Procedure:
- standard GoTaq-protocol
- template: colonie pcr clones of GA_K2201221
- primer fwd: Präfix_fwd
- primer rev: Suffix_rev
- annealing temperature: 56°C
- extension time: 40 seconds
- positive clone 2
multiplication of plasmids containing the Biobrick
Investigators: Yannic Kerkhoff
Superior experiment: preculture of the positive clone 2 of BBa_K2201221
Procedure:
- standard procedure
- 1,25µl Cam in 5mL LB
getting plasmids containing the Biobrick for further experiments
Investigators: Yannic Kerkhoff
Superior experiment: plasmid isolation of preculture of BBa_K2201200 clone 20
Procedure:
- standard protocol
- 240,6 ng/µL
getting plasmids containing the Biobrick for further experiments
Investigators: Yannic Kerkhoff
Superior experiment: plasmid isolation of preculture of BBa_K2201221 clone 2
Procedure:
- standard protocol
- 208,8 ng/µL
2017-06-26 - 2017-07-02
Check the sequence
Investigators: Christina Drake
Superior experiment: Sequenzingorder barnase
Procedure:
- concentration measurement by nanoprop: 177 ng/µl
- result: sequence was not barnase
Plasmidisolation and gibson assembly of pSB1C3-PlacUV5_PtNTT2 c30
Investigators: Camilla Maerz
Superior experiment: Construction of pSB1C3-PlacUV5_PtNTT2
Procedure:
- Plasmid DNA purification kit
- Nucleo spin plasmid protocol
- Gibson Assembly protocol
- transformation via heat shock protocol in E. coli DH5alpha
Colony PCR of T7RNAP from E. coli KRX
Investigators: Olga Schmidt
Superior experiment: Construction of the positive selection plasmid
Procedure:
- PCR with Grimson Taq polymerase (per reaction, 25 µL):
- 5 µL 5x Buffer
- 2.5 µL dNTPs (2 mM)
- 0.5 µL 3'-Primer (10 mM)
- 0.5 µL 5'-Primer (10 mM)
- 0.125 µL Grimson Taq polymerase
- 1 picked bacterial colony as template
- ad 25 µL H20
- Primer annealing: 64 °C
- Elongation: 150 s
- Agarose gel electrophoresis (1%)
- 100 V, 30 min
Isolate T7RNAP from the chromosome of KRX-e.coli
Investigators: Christina Drake
Superior experiment: PCR of T7RNAP from KRX-e.coli
Procedure:
- Q5 High-Fidelity PCR
- annealingtemparatur 64°C, elogationtime 1 min
- 1%-agarose-TAE-gel
- 1kb Marker Geneuler and 5 µl sample with 1 µl 6*Loading Dye
Plated out Barnase from strain collection
Investigators: Christina Drake
Superior experiment: Plated out Barnase from strain collection
Procedure:
- solved DNA in 100 µl SOC-medium
- plated out on chloramphenicolplate
- overnightincubation at 37°C
Colony PCR of pK18mobsacB-del_codA
Investigators: Camilla Maerz
Superior experiment: Construction of pK18mobsacB_codA_del
Procedure:
- Go Tag (Promega) protocol
- Primer: VR, VF2
- Primer annealing: 56 °C
- Elongation: 140 s
PCR of pSB1C3
Investigators: Olga Schmidt
Superior experiment: General
Procedure:
- PCR
- Q5 High-fidelily (NEB) protocol
- Denaturation: 64 °C
- Elongation: 60 s
- Agarose gel electrophoresis (1%)
- 100 V, 30 min
Gibson assembly of T7RNAP in pSB1C3
Investigators: Olga Schmidt
Superior experiment: Construction of the selection plasmid
Procedure:
- Gibson Assembly protocol
- transformation via heat shock protocol in E. coli BL21
Colony PCR of pSB1C3_T7RNAP c1-8
Investigators: Olga Schmidt
Superior experiment: Construction of the selection plasmid
Procedure:
- PCR
- Q5 High-fidelily (NEB) protocol
- Denaturation: 64 °C
- Elongation: 80 s
- Agarose gel electrophoresis (1%)
- 100 V, 30 min
Colony PCR of barnase (26.06.2017) c1&2
Investigators: Olga Schmidt
Superior experiment: Construction of selection plasmid
Procedure:
- PCR
- Go Tag (Promega) protocol
- Danaturation: 58 °C
- Elongation: 35 s
- Agarose gel electrophoresis (1%)
- 100 V, 30 min
2017-07-03 - 2017-07-09
Check the correct barnasegen
Investigators: Christina Drake
Superior experiment: PCR of barnase
Procedure:
- Go-Taq-protocol
- annealingtemparatur 58°C, elogationtime 30 s
- 1%-agarose-TAE-gel
- 100bp Marker Geneuler and 5 µl sample with 1 µl 6*Loading Dye
- result: PCR-product could not be barnase
2017-07-10 - 2017-07-16
Biobrick Assembly of 2B1
Investigators: Maximilian Edich
Superior experiment: Biobrick Construction with RuBisCo Parts
Procedure:
- BioBrick Assembly
- RuBisCoTAG mRFP as upstream part, T7-Terminator as downstream part, pSB1A3 as destination plasmid
- transformation via heat shock and DH5α Cells
- used 5µl from the ligation
- plated out on Amp plates and let them grow over night
Check the sequence
Investigators: Christina Drake
Superior experiment: Sequenzingorder barnase
Procedure:
- result: sequence was not barnase
- 3 ml LB-medium and 0,75 µl chloramphenicol
- inkubation overnight 37°C, 140rpm
Transformation of T7-Promotor
Investigators: Maximilian Edich
Superior experiment: Biobrick Construction with RuBisCo Parts
Procedure:
- transformation via electroporation of cells with T7-Promotor
Plasmidisolation of TyrRS-Heidelberg plasmid
Investigators: Olga Schmidt
Superior experiment: Construction of selection plasmid
Procedure:
- Plasmid DNA purification kit (Macherey-Nagel)
- Nucleo spin plasmid protocol
Preculture of colonies with T7-Promotor
Investigators: Maximilian Edich
Superior experiment: Biobrick Construction with RuBisCo Parts
Procedure:
- used 5µl LB media and kanamycin
2017-07-17 - 2017-07-23
Isolate the tRNA
Investigators: Christina Drake
Superior experiment: PCR of TyrRS-Heidelberg-plasmid
Procedure:
- plasmidisolation with MN protocol 5
- Q5 High-Fidelity PCR
- annealingtemparatur 53°C, elogationtime 20s
- Gibbsonassembly with 5µl backbone and 0.5 µl insert
- transformation via heat shock
- overnight incubation at 37°C
Isolation of T7-Promotor
Investigators: Laura Schlueter
Superior experiment: Biobrick Construction with RuBisCo Parts
Procedure:
- Nucleospin Plasmidisolation protocol
- got four products with the concentrations 50.1 ng/µl, 16.3 ng/µl, 24,2 ng/µl and 2.1 ng/µl
Colony PCR with 2B1 Colonies
Investigators: Maximilian Edich
Superior experiment: Biobrick Construction with RuBisCo Parts
Procedure:
Biobrickassembly of 2B2 to 2B8, B1 and A1
Investigators: Maximilian Edich
Superior experiment: Biobrick Construction with RuBisCo Parts
Procedure:
- BioBrick Assembly
- digest and ligate RuBisCo parts as upstreampart and T7-Terminator/Promotor as downstreampart and pSB1A3 as destination plasmid
- ligate TAG2 and T7-Terminator to 2B2
- ligate TAG111 and T7-Terminator to 2B3
- ligate TAG474 and T7-Terminator to 2B4
- ligate TAG2+TAG111 and T7-Terminator to 2B5
- ligate TAG2+TAG474 and T7-Terminator to 2B6
- ligate TAG111+TAG474 and T7-Terminator to 2B7
- ligate TAG2+TAG111+TAG474 and T7-Terminator to 2B8
- ligate RuBisCo mRFP and T7-Terminator to B1
- ligate Carboxysome and T7-Promotor to A1
- preculture of positive 2B1 colony
Transformation of test devices
Investigators: Camilla Maerz
Superior experiment: Interlab study
Procedure:
- dilute test devives (distribution plate 6 & 7) in 10 µL ddH20
- Transformation
- 1 µL plasmid from each plate
- transformation via heat shock protocol in E. coli XL1-blue
Transformation of 2B2-2B8, B1 and A1
Investigators: Maximilian Edich
Superior experiment: Biobrick Construction with RuBisCo Parts
Procedure:
Plasmidisolation of 2B1
Investigators: Maximilian Edich
Superior experiment: Biobrick Construction with RuBisCo Parts
Procedure:
- Nucleospin Plasmidisolation protocol
- got 47 ng/µl and 41 ng/µl
Reverse the insert
Investigators: Christina Drake
Superior experiment: PCR of T7GFP
Procedure:
- Q5 High-Fidelity PCR
- Insert: annealingtemparatur 60°C,
- Backbone:elogationtime 30s: annealingtemparatur 60°C, elogationtime 1 min
- 1%-agarose-TAE-gel
- result: PCR didn`t work
Reverse the insert
Investigators: Christina Drake
Superior experiment: PCR of T7GFP
Procedure:
- 1%-agarose-TAE-gel
- result: PCR did work
- Gibson Assembly
- transformation via heat shock
PCR of BBa_I746909 and pSB1C3
Investigators: Olga Schmidt
Superior experiment: Integration of CDS of BBa_I746909 in pSB1C3
Procedure:
- PCR of I746909
- Q5 High-fidelily (NEB) protocol
- Primer: 17lk & 17lj
- Denaturation: 60 °C
- Elongation: 40 s
- PCR of pSB1C3
- Q5 High-fidelily (NEB) protocol
- Primer: 17ll & 17li
- Denaturation: 60 °C
- Elongation: 70 s
- Agarose gel electrophoresis (1%)
- 100 V, 30 min
Preparation of over-night culture
Investigators: Camilla Maerz
Superior experiment: Interlab study
Procedure:
- Over-night culture of negative control, positive control, test device 1-6
- LB-media + Cm were inoculated
- Cultures were incubated over night (37°C, 200 rpm)
2017-07-24 - 2017-07-30
Plasmidisolation of pSB1C3_I746909
Investigators: Olga Schmidt
Superior experiment: Integration of CDS of BBa_I746909 in pSB1C3
Procedure:
- Plasmid DNA purification kit (Macherey-Nagel)
- Nucleo spin plasmid protocol
Repeat the degestion and transformation from the 18th and 19th july
Investigators: Maximilian Edich
Superior experiment: Biobrick Construction with RuBisCo Parts
Procedure:
Cellgrowth with the plasmid
Investigators: Christina Drake
Superior experiment: Overnightculture of tRNA-psB1C3
Procedure:
- 3 ml LB-medium and 0,75 µl chloramphenicol
- inkubation overnight 37°C, 140rpm
Cloning of the ori (pSB6A1) in pSB1C3
Investigators: Laura Schlueter
Superior experiment: aaRS Plasmid (without aaRS)
Procedure:
- Gibson Assembly Master Mix
- Backbone: 3.28 µL Ori 1.0: 1.73 µL
- Backbone: 0.32 µL Ori 1.1: 4.68 µL
- Backbone: 0.90 µL Ori 3.0: 4.10 µL
- Trafo: heatshock
- not successfull because of the wrong overlapps (try again)
Colony PCR of the Retrafo of pRS (because of mixed culture)
Investigators: Laura Schlueter
Superior experiment: aaRS plasmid
Procedure:
- Sequencing was not succesfull (maybe mixed colony), so I did a retrafo with the aim of a successful sequencing (incorporation of the aaRS in pSB1C3)
- pRS 3: 1-5
- pRS 4: 1-5
- pRS 5: 1-5
- pRS 7: 1-5
- no positive result (no band at 1000 bp)
Amplification of pSB1C3 for the cloning of the ori in pSB1C3
Investigators: Laura Schlueter
Superior experiment: aaRS plasmid
Procedure:
- PCR with Q5 High-Fidelity Polymerase Master Mix, Primer jb,jn, 65°C
- gelelectrophoresis, 1% Agarose
- band 2000-2500 bp (supposed to be around 2200 bp)
- Clean up from the gel
- pSB1C3(nur ori): 22.2 ng/µL
Amplification of pSB1C3 fragments for a 4 part Gibson (see page 4 in the paper-labbook)
Investigators: Laura Schlueter
Superior experiment: aaRS plasmid
Procedure:
- PCR, Q5 Master Mix
- Primer jj, jn for fragment 2, 65°C
- Primer jb, ht for fragment 4, 65°C
- gelelectrophoresis, 1% Agarose
- band at 300 (fragment 2, supposed to be around 326 bp)
- band at 1200 (fragment 4, supposed to be around 1233 bp)
- Clean up from the gel
- fragment 2: 18.5 ng/µL (overlapp(aaRS)_pSB1C3_overlapp(ori))
- fragment 4: 19.1 ng/µL (overlapp(ori)_pSB1C3_overlapp(aaRS))
Colony PCR of 2B2-2B8, B1 and A1
Investigators: Maximilian Edich
Superior experiment: Biobrick Construction with RuBisCo Parts
Procedure:
- GoTaq® G2 PCR
- most have negative results -> create new destination plasmid pSB1A3
Colony PCR of retrafo and more colonies of the pRS
Investigators: Laura Schlueter
Superior experiment: aaRS plasmid
Procedure:
- GoTaq Master Mix, Primer vr,vf
- 2 strong bands at 400 bp and 600 bp, small bands of pRS3 6 and pRS4 9 at around 1200-1500 bp
- bands of pRS31, pRS32, pRS34, pRS36, pRS39 at around 1200-1500 bp
- GoTaq Master Mix, Primer hq, jk (Gibson Assembly primer) with the colonies pRS31, pRS32, pRS34, pRS36, pRS39
- pRS31, pRS32, pRS39 : strong bands at around 1200 bp
- the two positive colony PCRs (vf-vr, hq-jk) for these probes, successful integration of the Tyr-aaRS in pSB1C3 seems to be successfull
- pRS31, pRS32, pRS39 send to sequencing
pRS31, pRS32, pRS39 plasmid isolation
Investigators: Laura Schlueter
Superior experiment: aaRS plasmid
Procedure:
- Macherey-Nagel purification kit
- pRS31: 324.1 ng/µL
- pRS32: 231.8 ng/µL
- pRS39: 225.5 ng/µL
- send tosequencing
- pRS31, pRS39 have the pSB1C3 with the Tyr-aaRS incorporated (but on position 32, the Arginine (cgt) should be changed to an Leucine (ctg), but it is not. On position 290 there really is a missing Leu, on position 268 the Asparagin is changed to an Arginine)
2017-07-31 - 2017-08-06
construct a biobrick
Investigators: Christina Drake
Superior experiment: Aquacloning of tRNA and backbone
Procedure:
- transformation via heatschock
plasmid isolation of the positive colonies of the retrafo pRS4 4 (wrong!) and pRS3 12
Investigators: Laura Schlueter
Superior experiment: aaRS plasmid
Procedure:
- Macherey-Nagel purification kit with each 3 mL culture
- pRS4 4: 44 ng/µL
- pRS3 12: 68.3 ng/µL
- not send to sequencing jet
4 part Gibson and Trafo
Investigators: Laura Schlueter
Superior experiment: aaRS plasmid
Procedure:
- Gibson Assembly Master Mix with 5 µl template
- pSB1C3 fragment (2): 18.5 ng/µL around 350 bp 2.2 µL
- pSB1C3 fragment (4): 19.1 ng/µL around 1300 bp 0.8 µL
- oK1.1 (pMB1 from pSB6A1): 5.8 ng/µL around 1300 bp 1.8 µL
- Tyr-aaRS: 68.5 ng/µL around 1000 bp 0.2 µL
- Trafo via heatshock
Gibson of the ori in pSB1C3 (pori) and Trafo
Investigators: Laura Schlueter
Superior experiment: aaRS plasmid
Procedure:
- Gibson Assembly Master Mix with 5 µL template
- pSB1C3(nur ori): 22.2 ng/µL around 2100 bp 1.57 µL
- ok1.0: 5.80 ng/µL around 1258 bp 3.43 µL
- Trafo via heatshock
Transfer positive colonies
Investigators: Maximilian Edich
Superior experiment: Biobrick Construction with RuBisCo Parts
Procedure:
- picked and transfered positiv colonies of 2B2, 2B6, 2B7, 2B8 and A1 onto a new plate
- incubate over night
- 2B2 and 2B8 are contaminated with negativ colonies
construct a biobrick
Investigators: Christina Drake
Superior experiment: Aquacloning of tRNA and backbone
Procedure:
- transformation via heatschock
construct a biobrick
Investigators: Christina Drake
Superior experiment: Gibbsonassembly of barnase and backbone
Procedure:
- transformation via heatschock
Preculture of 2B6, 2B7 and A1
Investigators: Maximilian Edich
Superior experiment: Biobrick Construction with RuBisCo Parts
Procedure:
- used 3µl LB media
Cellgrowth with the plasmid
Investigators: Christina Drake
Superior experiment: Overnightculture of tRNA-psB1C3 and barnase-psB1C3
Procedure:
- 3 ml LB-medium and 0,75 µl chloramphenicol
- inkubation overnight 37°C, 140rpm
Plasmidisolation
Investigators: Maximilian Edich
Superior experiment: Biobrick Construction with RuBisCo Parts
Procedure:
- Nucleospin Plasmidisolation protocol
- got 67.3 ng/µl of 2B6
- got 32.7 ng/µl of 2B7
- got 51.6 ng/µl of A1
To get and purified the plasmid
Investigators: Christina Drake
Superior experiment: Plasmidisolation of barnase-plasmid and tRNA-plasmids
Procedure:
- protocol from Macherley-Nagel: Plasmid DNA purification, protocol 5: NucleoSpin
- concentration measurement by nanoprop
- sequenzingorder
Redo digestion of the destination plasmid
Investigators: Maximilian Edich
Superior experiment: Biobrick Construction with RuBisCo Parts
Procedure:
- analyze the destination plasmid digestion via gelelectrophoresis
- pSB1A3 digestion was not effective
- redo the digestion and analyze it on the gel
- new dgestion was very effective
2017-08-07 - 2017-08-13
Amplification of the backbone (pSB1C3) and insert (Tyr-aaRS)
Investigators: Laura Schlueter
Superior experiment: aaRS plasmid
Procedure:
- PCR Q5 Polymerase (no Master Mix)
- for 50 µL reaction
- 10 µL Reaction Buffer
- 5 µL 2mM dNTPs
- 1 µL Template
- 0,5 µL High Fidelity DNA Polymerase
- 10.0 µL High GC Enhancer
- 18.5 µL ddest H2O
- 5.0 µL Primer
- pSB1C3 (from RuBisCo, ncAA team): ht, jk 65°C
- aaRS (from Heidelberg) : hq, jk 65°C (mistake! should be 58°C, redone identically with 58°C)
- digestion of the PCR products with Dpn1: 5 µL CutSmart Reaktionsbuffer and 1 µL Dpn1 per 50 µL PCR product, on 37°C over night)
- agarose-gelelectrophhoresis, 1% (bands of the right size)
- No further use, because results of the sequencing which show that the integration of the incorporation of the Tyr-aaRS in pSB1C3 was already successful (30.06.2017)
Ligation of 2B2-2B5 and B1
Investigators: Maximilian Edich
Superior experiment: Biobrick Construction with RuBisCo Parts
Procedure:
- Ligation protocol from the BioBrick Assembly
- Variation: incubated 1h at RT instead of 10min
characterisation of T7GFP revers
Investigators: Christina Drake
Superior experiment: preparation of KRX-competent cells
Procedure:
- heatschocktransformation of T7GFP and T7GFP revers in KRX
construct a biobrick for the selectionplasmid
Investigators: Christina Drake
Superior experiment: PCR of T7RNA-Polymerase
Procedure:
- 1%-agarose-TAE-gel
- result: PCR did work
- Gibson Assembly
- transformation via heat shock
Amplification of the backbone (pRS31) and insert (ori from pSB6A1, oK1.0) for Gibson assembly
Investigators: Laura Schlueter
Superior experiment: aaRS plasmid
Procedure:
- PCR Q5 Polymerase (protocoll of 07.08.2017)
- pRS31: primer jb, jn 65°C
- oK1: primer jl, jc 65°C
- digestion with Dpn1 (protocoll of 07.08.2017)
- agarose-gelelectrophhoresis, 1%
- band around 1200 bp (oK1.1) / 2500 bp (pRS31), positive
Gibson Assembly and Trafo of the pRS31 and oK1.0
Investigators: Laura Schlueter
Superior experiment: aaRS plasmid
Procedure:
- agarose-gelelectrophhoresis, 1%
- purification from the gel with the Macherey-Nagel purification kit
- oK1.1: 22.5 ng/µL
- pRS32: 9.5 ng/µL
- Gibson Assembly Master Mix with:
- 4.1 µL of oK1.1: around 2480 bp 9.5 ng/µL
- 0.9 µL of pRS32: around 1250 bp 22.5 ng/µL
- Trafo via heatshock
getting positive clones for further work
Investigators: Yannic Kerkhoff
Superior experiment: heatshock transformation of assembled fragments F13 and NPA-RS gene synthesis
Procedure:
- standard heatshock protocol
- used own produced chemo competend DH5alpha
- streaked out on LB-plates with Cam
multiplication, verification and purification of the needed fragment F13: linearized ONBY-Part with removal of the ONBY-RS
Investigators: Yannic Kerkhoff
Superior experiment: Q5-PCR, gel and PCR-cleanUp of F13
Procedure:
- standard Q5-PCR protocol
- template: K1416000
- primer fwd: 17hl
- primer rev: 17mv
- annealing temperature: 63°C
- extension time: 90 seconds
- standard PCR-cleanUp protocol
- 80,6 ng/µL
Assemble the fragments to get the Part BBa_K2201200
Investigators: Yannic Kerkhoff
Superior experiment: Gibson assembly of F13 and NPA-RS gene synthesis
Procedure:
- standard Gibson Assembly protocol
getting positive clones for further work
Investigators: Yannic Kerkhoff
Superior experiment: heatshock transformation of assembled fragments F5,F1,F3
Procedure:
- standard heatshock protocol
- used own produced chemo competend DH5alpha
- streaked out on LB-plates with Cam
multiplication, verification and purification of the needed fragment F1: GFPLinker1
Investigators: Yannic Kerkhoff
Superior experiment: Q5-PCR, gel and PCR-cleanUp of F1
Procedure:
- standard Q5-PCR protocol
- template: BBa_E0400
- primer fwd: 17gk
- primer rev: 17gm
- annealing temperature: 58°C
- extension time: 50 seconds
- standard PCR-cleanUp protocol
- 15,9 ng/µL
multiplication, verification and purification of the needed fragment F3: StrepLinker1
Investigators: Yannic Kerkhoff
Superior experiment: Q5-PCR, gel and PCR-cleanUp of F3
Procedure:
- standard Q5-PCR protocol
- template: BBa_KJ36848
- primer fwd: 17go
- primer rev: 17gq
- annealing temperature: 61°C
- extension time: 30 seconds
- standard PCR-cleanUp protocol
- 17,9 ng/µL
Biobrickassembly
Investigators: Maximilian Edich
Superior experiment: Biobrick Construction with RuBisCo Parts
Procedure:
- BioBrick Assembly
- repeat digestion of T7-Terminator downstrampart
- ligate over night
Transformation of new plasmids
Investigators: Maximilian Edich
Superior experiment: Biobrick Construction with RuBisCo Parts
Procedure:
multiplication, verification and purification of the needed fragment F15: linear GLS1
Investigators: Yannic Kerkhoff
Superior experiment: Q5-PCR, gel and PCR-cleanUp of F15
Procedure:
- standard Q5-PCR protocol
- template: P2: GLS2
- primer fwd: 17go
- primer rev: 17gm
- annealing temperature: 60°C
- extension time: 190 seconds
- standard PCR-cleanUp protocol
- 36,9 ng/µL
Primer annealing preparation
Investigators: Camilla Maerz
Superior experiment: M.A.X restriction system - Primer annealing test
Procedure:
- Both Primers (17jh; 17ji) were resuspended in 1x Composite Buffer
- Dilutions: 100 µM, 10 µM, 5 µM, 2.5 µM, 0.5 µM
- OD 260 were measured to determine the real concentration
2017-08-14 - 2017-08-20
Screening for positive transformations containing the Biobrick
Investigators: Yannic Kerkhoff
Superior experiment: GoTaq PCR of 4 clones of BBa_K2201220
Procedure:
- standard GoTaq-protocol
- template: colonie pcr clones of GA_K2201220
- primer fwd: Präfix_fwd
- primer rev: Suffix_rev
- annealing temperature: 56°C
- extension time: 40 seconds
- positive clone 3
multiplication of plasmids containing the Biobrick
Investigators: Yannic Kerkhoff
Superior experiment: preculture of the positive clone 3 of BBa_K2201220
Procedure:
- standard procedure
- 1,25µl Cam in 5mL LB
Primer annealing of 17jh & 17ji
Investigators: Camilla Maerz
Superior experiment: M.A.X restriction system - Primer annealing test
Procedure:
- Protocol for annealing oligonucleotides (sigmaaldrich)
- Eqimolar Primers were mixed (50 µL)
- Thermal profile (thermocycler)
- 95 °C, 2 min
- Cool to 25 °C over 45 min
- Cool to 4 °C for temporary storage
Integration of barnase in pSB1C3
Investigators: Olga Schmidt
Superior experiment: Construction of selection plasmid
Procedure:
- Restriction Digest of pSB1C3_I749606
- 5 µL 10x Universal buffer
- 5.54 µL plasmid
- 1µL XbaI
- 1µL PstI
- filled up to final volume 50 µL with ddH20
- Incubation: 30 min, 37 °C
- Heatinactivation: 20 min, 80 °C
- Agarose gel electrophoresis (1%)
- 100 V, 30 min
- PCR and DNA purification kit (Macherey-Nagel)
- Nucleo spin PCR and Monarch® PCR & DNA Clean-up protocol
- Gibson Assembly protocol
- transformation via heat shock protocol in E. coli DH5alpha
- transformation via electroporation protocol in E. coli DH5alpha
PCR of T7RNAP_BL
Investigators: Olga Schmidt
Superior experiment: Construction of the selection plasmid
Procedure:
- PCR
- Go Tag (Promega) protocol
- Danaturation: 56 °C
- Elongation: 180 s
- Agarose gel electrophoresis (1%)
- 100 V, 30 min
getting plasmids containing the Biobrick for further experiments
Investigators: Yannic Kerkhoff
Superior experiment: plasmid isolation of preculture of BBa_K2201220 clone 3
Procedure:
- standard protocol
- 108,9 ng/µL
Combination of two biobricks to get the composite part BBa_K2201320 for protein expression
Investigators: Yannic Kerkhoff
Superior experiment: Biobrick Assembly of BBa_K608006 and BBa_K2201220
Procedure:
- Standard BioBrick Assembly protocol
- Backbone: BBa_J04450, 7,7µL
- upstream: BBa_K608006, 3,6µL
- downstram: BBa_K2201220, 5,0µL
- Ligation time was 12 h at 8 °C
Colony and plasmid PCR of T7RNAP_BL c1-4
Investigators: Olga Schmidt
Superior experiment: Construction of the selection plasmid
Procedure:
- PCR
- Go Tag (Promega) protocol
- Primer: VR & VF
- Danaturation: 56 °C
- Elongation: 180 s
- Agarose gel electrophoresis (1%)
- 100 V, 30 min
getting positive clones for further work
Investigators: Yannic Kerkhoff
Superior experiment: heatshock transformation of the Biobrick Assembly product BBa_K2201320
Procedure:
- standard heatshock protocol
- used own produced chemo competend BL21(DE3)
- streaked out on LB-plates with Cam
Colony PCR of pSB1C3_barnase (gblock) c1-70
Investigators: Olga Schmidt
Superior experiment: Construction of selection plasmid
Procedure:
- PCR
- Go Tag (Promega) protocol
- Primer: Vr & VF2
- Danaturation: 56.5 °C
- Elongation: 40 s
- Agarose gel electrophoresis (1%)
- 100 V, 30 min
Screening for positive transformations containing the Biobrick
Investigators: Yannic Kerkhoff
Superior experiment: GoTaq PCR of 19 clones of BBa_K2201320
Procedure:
- standard GoTaq-protocol
- template: colonie pcr clones of GA_K2201320
- primer fwd: T7-eva_fwd
- primer rev: Suffix_rev
- annealing temperature: 51°C
- extension time: 80 seconds
- positive clone 19
multiplication of plasmids containing the Biobrick
Investigators: Yannic Kerkhoff
Superior experiment: preculture of the positive clone 19 of BBa_K2201320
Procedure:
- standard procedure
- 1,25µl Cam in 5mL LB
Combination of two biobricks to get the composite part BBa_K2201321 for protein expression
Investigators: Yannic Kerkhoff
Superior experiment: Biobrick Assembly of BBa_K608006 and BBa_K2201221
Procedure:
- Standard BioBrick Assembly protocol
- Backbone: BBa_J04450, 2,0µL
- upstream: BBa_K608006, 3,6µL
- downstram: BBa_K2201221, 2,4µL
- Ligation time was 12 h at 8°C
Primer annealing of 17jl & 17jm
Investigators: Camilla Maerz
Superior experiment: M.A.X restriction system - Primer annealing test
Procedure:
- Both Primers were resuspended in ddH20 to a final concentration of 100 µM
- HEPES annealing standard protocol
- Aqua annealing standard protocol
- Final volume: 20 µL
- Agarose gel electrophoresis (2%)
- 60 V, 50 min
multiplication of plasmids containing the Biobrick
Investigators: Yannic Kerkhoff
Superior experiment: preculture of BL21(DE3) BBa_K2201320
Procedure:
- standard procedure
- 1,25µl Cam in 10mL LB
- 12 h at 37°C
expression of the fusion protein
Investigators: Yannic Kerkhoff
Superior experiment: preculture of BL21(DE3) BBa_K2201320 with IPTG
Procedure:
- standard procedure
- 1,25µl Cam in 10mL LB
- 12 h at 37°C
- induced with IPTG 0.8mM
getting plasmids containing the Biobrick for further experiments
Investigators: Yannic Kerkhoff
Superior experiment: plasmid isolation of preculture of BBa_K2201320 clone 19
Procedure:
- standard protocol
- 454,4 ng/µL
multiplication of plasmids containing the Biobrick
Investigators: Yannic Kerkhoff
Superior experiment: preculture of BL21(DE3) BBa_K2201320
Procedure:
- standard procedure
- 1,25µl Cam in 10mL LB
- 12 h at 37°C
expression of the fusion protein
Investigators: Yannic Kerkhoff
Superior experiment: preculture of BL21(DE3) BBa_K2201320 with IPTG
Procedure:
- standard procedure
- 1,25µl Cam in 10mL LB
- 12 h at 37°C
Ligation of pSB1C3 and barnase (gblock)
Investigators: Olga Schmidt
Superior experiment: Construction of selection plasmid
Procedure:
- Ligation reaction
- 7 µL vector
- 3 µL gblock
- 2 µL T4 DNA ligase buffer
- 1 µL T4 DNA ligase
- 17 µL ddH20
- Incubation: RT, 1 h
Colony PCR of pSB1C3_barnaseBL c1-60
Investigators: Olga Schmidt
Superior experiment: Construction of selection plasmid
Procedure:
- PCR
- Q5 High-fidelily (NEB) protocol
- Denaturation: 56.5 °C
- Elongation: 45 s
- Agarose gel electrophoresis (1%)
- 100 V, 30 min
getting positive clones for further work
Investigators: Yannic Kerkhoff
Superior experiment: heatshock transformation of the Biobrick Assembly product BBa_K2201321
Procedure:
- standard heatshock protocol
- used own produced chemo competend BL21(DE3)
- streaked out on LB-plates with Cam
- standard cultivation protocoll
Investigators: Yannic Kerkhoff
Superior experiment: Cultivation of BL21(DE3) with BBa_K2201320
Procedure:
- 2x 100mL LB-media with 25µL Cam in 1000mL flasks
- innoculation with 3.3 mL preculture of OD 3 to get a starting OD of 0.1
- incubation for four hours to a OD of 0.6
- addition of 500µL 0.5mM IPTG each
- incubation for 8 hours at 37°C and 24 hours at 18°C
- harveting of the cells
Native PAGE of annealed primers (17jl & 17jm)
Investigators: Camilla Maerz
Superior experiment: M.A.X restriction system - Primer annealing test
Procedure:
- Native DNA PAGE standard protocol
- tested samples:
- HEPES annealing (17jl & 17jm): 100 µM, 10 µM, 5 µM, 2.5 µM, 1 µM, 0.5 µM
- Aqua annealing (17jl & 17jm): 100 µM, 10 µM, 5 µM, 2.5 µM, 1 µM, 0.5 µM
- Variation: ~40 mA
2017-08-21 - 2017-08-27
Native PAGE of annealed primers (17jl & 17jm)
Investigators: Camilla Maerz
Superior experiment: M.A.X restriction system - Primer annealing test
Procedure:
- Native DNA PAGE standard protocol
- tested samples:
- HEPES annealing (17jl & 17jm): 100 µM, 10 µM, 5 µM, 2.5 µM, 1 µM, 0.5 µM
- Aqua annealing (17jl & 17jm): 100 µM, 10 µM, 5 µM, 2.5 µM, 1 µM, 0.5 µM
- Variation: 100 V, 1 h
Plasmidisolation of pSB1C3_barnaseB c5, 22, 30, 31, 32, 45
Investigators: Olga Schmidt
Superior experiment: Construction of selection plasmid
Procedure:
- Plasmid DNA purification kit (Zymo PURE)
- Nucleo spin plasmid protocol
PCR of KanR & pSB1C3
Investigators: Olga Schmidt
Superior experiment: Construction of selection plasmid
Procedure:
- PCR
- Q5 High-fidelily (NEB) protocol
- Primer: 17nx & 17ny (Kan)
- Primer: 17od & 17oe (pSB1C3)
- Denaturation: 54 °C & 60 °C
- Elongation: 30 s & 75 s
- Restriction Digest
- 5 µL 10x CutSmart buffer
- 1 µg plasmid
- 1 µL DpnI
- filled up to final volume 50 µL with ddH20
- Incubation: ON, 37 °C
- Inactivation: 20 min, 80 °C
multiplication of plasmids containing the Biobrick
Investigators: Yannic Kerkhoff
Superior experiment: preculture of the positive clone 3 of BBa_K2201200
Procedure:
- standard procedure
- 1,25µl Cam in 5mL LB
Restriction digest of KanR fragment
Investigators: Olga Schmidt
Superior experiment: Construction of selection plasmid
Procedure:
- Restriction Digest
- 5 µL NEB2.1 buffer
- 2 µL DNA
- 1 µL XbaI
- 1 µL PstI
- filled up to final volume 50 µL with ddH20
- Incubation: 60 min, 37 °C
- Heatinactivation: 20 min, 80 °C
Aim of the Experiment
Investigators: Yannic Kerkhoff
Superior experiment: GoTaq PCR of 32 clones of BBa_K2201200
Procedure:
- standard GoTaq-protocol
- template: colonie pcr clones of GA_K2201200
- primer fwd: 17ns
- primer rev: 17nt
- annealing temperature: 56°C
- extension time: 40 seconds
- positive clone 20
Native PAGE of annealed primers (17jl & 17jm)
Investigators: Camilla Maerz
Superior experiment: M.A.X restriction system - Primer annealing test
Procedure:
- Native DNA PAGE standard protocol
- tested samples:
- HEPES annealing (17jl & 17jm): 100 µM, 10 µM, 5 µM, 2.5 µM, 1 µM, 0.5 µM
- Aqua annealing (17jl & 17jm): 100 µM, 10 µM, 5 µM, 2.5 µM, 1 µM, 0.5 µM
- Variation: 60 V, 2 h
multiplication of plasmids containing the Biobrick
Investigators: Yannic Kerkhoff
Superior experiment: preculture of BL21(DE3) BBa_K2201321
Procedure:
- standard procedure
- 1,25µl Cam in 5mL LB
getting plasmids containing the Biobrick for further experiments
Investigators: Yannic Kerkhoff
Superior experiment: plasmid isolation of preculture of BBa_K2201321 clone 3
Procedure:
- standard protocol
- 319,1 ng/µL
Native Page of annealed primers (17jl & 17jm; 17jh & 17ji)
Investigators: Camilla Maerz
Superior experiment: M.A.X restriction system - Primer annealing test
Procedure:
- Native DNA PAGE standard protocol
- tested samples:
- HEPES annealing (17jl & 17jm): 0.5 µM
- Aqua annealing (17jl & 17jm): 0.5 µM
- Composite Buffer annealing (17jh & 17 jm): 0.5 µM
- all Primers (ssDNA): 1 µM
2017-08-28 - 2017-09-03
Native PAGE of annealed primers (HEPES annealing & Composite Buffer annealing) of the same concentration to test if all annealings have the same quality
Investigators: Camilla Maerz
Superior experiment: M.A.X restriction system - Primer annealing test
Procedure:
- all samples were diluted to a final concentration of 0.5 µM
- Native DNA PAGE standard protocol
getting positive clones for further work
Investigators: Yannic Kerkhoff
Superior experiment: heatshock transformation of the Biobrick Assembly product CP3
Procedure:
- standard heatshock protocol
- used own produced chemo competend BL21(DE3)
- streaked out on LB-plates with Cam
Restriction digest of control annealings (mutC)
Investigators: Camilla Maerz
Superior experiment: M.A.X restriction system
Procedure:
- Restriction Digest (MnlI) of aqua annealing sample: mutC (0.5 µM)
- 2 µL MnlI
- 5 µL CutSmart
- 25 µL mutC dsDNA
- 18 µL nuclease-free H2O
- Incubation: 37 °C, 1 h
- Inactivation: 20 min., 65 °C
- Native DNA PAGE standard protocol
- Tested samples:
- digested mutC sample
- native mutC sample
- ssDNA primers (17jl & 17jm)
GoTaq PCR of 14 clones of BBa_K2201322
Investigators: Yannic Kerkhoff
Superior experiment: Screening for positive transformations containing the Biobrick
Procedure:
- standard GoTaq-protocol
- template: colonie pcr clones of BBA_K2201322
- primer fwd: Präfix_fwd
- primer rev: Suffix_rev
- annealing temperature: 56°C
- extension time: 40 seconds
- positive: clone 3, 4, 5
Restriction digest of aqua & HEPES annealing
Investigators: Camilla Maerz
Superior experiment: M.A.X restriction system
Procedure:
- Restriction Digest (MnlI) of aqua & HEPES annealing sample: mutC (0.5 µM)
- 2 µL MnlI
- 5 µL CutSmart
- 25 µL mutC dsDNA
- 18 µL nuclease-free H2O
- Incubation: 37 °C, 1 h
- Inactivation: 20 min., 65 °C
- Native DNA PAGE standard protocol
- Tested samples:
- digested mutC sample (aqua & HEPES annealing)
- native mutC sample
- ssDNA primers (17jl & 17jm)
Aqua annealing of mutA, mutT, mutG and mutC
Investigators: Camilla Maerz
Superior experiment: M.A.X restriction system
Procedure:
- Aqua annealing standard protocol (final concentration: 0.5 µM)
- mutA: 17jf & 17jg
- mutT: 17jj & 17jk
- mutG: 17jh & 17ji
- mutC: 17jl & 17jm
- final volume of each sample: 50 µL
2017-09-04 - 2017-09-10
Restriction digest of control annealings (mutA, mutT, mutG, mutC) and aqua annealing of oligo2
Investigators: Camilla Maerz
Superior experiment: M.A.X restriction system
Procedure:
- Oligo2 annealing
- Aqua annealing standard protocol (final concentration: 0.5 µM)
- Restricted samples:
- MnlI -> mutC, oligo2
- SapI -> mutG, oligo2
- BsaI -> mutT, oligo2
- EciI -> mutA, oligo2
- Restriction Digest:
- 1 µL Enzyme
- 5 µL CutSmart
- 25 µL mutC dsDNA
- 19 µL nuclease-free H2O
- Incubation: 37 °C, 1 h
- Inactivation: 20 min., 65 °C
- Native DNA PAGE standard protocol
SDS-Page and western blot of BBa_K2201321
Investigators: Yannic Kerkhoff
Superior experiment: SDS-Page and western blot of BBa_K2201321
Procedure:
- standard SDS protocoll
- standard Western blot protocoll
integration of the mRFP (BBa_J04450) in the aaRS plasmid as optical controle for the integration of an randomerized dsDNA
Investigators: Laura Schlueter
Superior experiment: aaRS plasmid
Procedure:
- amplification of the mRFP (Phusion Master Mix), Primer vg, vh, annealing temperature: 60°C
- amplification of the library backbone(Phusion Master Mix), Primer 17ve, 17vf, Annealing temperature: 61°C
- Gibson Assembly with Gibson Master Mix + mRFP (3.1 µL) + library backbone (1.9 µL)
- transformation in DH5a via heatshock
Generating the randomerized dsDNA for the library
Investigators: Laura Schlueter
Superior experiment: aaRS plasmid
Procedure:
- ssDNA Annealing protocoll with the primers 17vi (1 µL), 17vj (1,4 µL)
- agarose gelelectrophoresis, 3 %, positive result: bands of 120 bp
Generating the library
Investigators: Laura Schlueter
Superior experiment: aaRS plasmid
Procedure:
- Gibson Assembly Master Mix + dsDNA (3.5 µl) + library Plasmid (0.5 µL) with the optical controle
- transformation in DH5a via heatshock
2017-09-11 - 2017-09-17
Annealing, restriction digest and native PAGE of all samples
Investigators: Camilla Maerz
Superior experiment: M.A.X restriction system
Procedure:
- Aqua annealing standard protocol (final concentration: 0.5 µM)
- mutA: 17jf & 17jg
- mutT: 17jj & 17jk
- mutG: 17jh & 17ji
- mutC: 17jl & 17jm
- oligo2: olgio2_sense & oligo2_antisense
- final volume of each sample: 50 µL
- Restricted samples:
- MnlI -> mutC, oligo2
- SapI -> mutG, oligo2
- BsaI -> mutT, oligo2
- EciI -> mutA, oligo2
- Restriction Digest:
- 1 µL Enzyme
- 5 µL CutSmart
- 25 µL mutC dsDNA
- 19 µL nuclease-free H2O
- Incubation: 37 °C, 1 h
- Inactivation: 20 min., 65 °C
- Native DNA PAGE standard protocol
Generating the library
Investigators: Laura Schlueter
Superior experiment: aaRS plasmid
Procedure:
- Gibson Assembly Master Mix + dsDNA (3.5 µl) + library Plasmid (0.5 µL) with the optical controle (8 x)
- transformation in DH5a via heatshock (8 x)
Generating the library
Investigators: Laura Schlueter
Superior experiment: aaRS plasmid
Procedure:
- Gibson Assembly Master Mix + dsDNA (3.5 µl) + library Plasmid (0.5 µL) with the opotical controle (16 x)
- transformation in DH5a via heatshock (16 x)
Generating the library
Investigators: Laura Schlueter
Superior experiment: aaRS plasmid
Procedure:
- Gibson Assembly Master Mix + dsDNA (3.5 µl) + library Plasmid (0.5 µL) with the opotical controle (25x)
- transformation in DH5a via heatshock (25 x)
Generating the library
Investigators: Laura Schlueter
Superior experiment: aaRS plasmid
Procedure:
- Gibson Assembly Master Mix + dsDNA (3.5 µl) + library Plasmid (0.5 µL) with the opotical controle (25x)
- transformation in DH5a via heatshock (25 x)
Generating the library
Investigators: Laura Schlueter
Superior experiment: aaRS plasmid
Procedure:
- Gibson Assembly Master Mix + dsDNA (3.5 µl) + library Plasmid (0.5 µL) with the opotical controle (25x)
- transformation in DH5a via heatshock (25 x)
Annealing, restriction digest and native PAGE of all samples
Investigators: Camilla Maerz
Superior experiment: M.A.X restriction system
Procedure:
- Aqua annealing standard protocol (final concentration: 0.5 µM)
- mutA: 17jf & 17jg
- mutT: 17jj & 17jk
- mutG: 17jh & 17ji
- mutC: 17jl & 17jm
- oligo2: olgio2_sense & oligo2_antisense
- final volume of each sample: 2x50 µL
- Restricted samples:
- MnlI -> mutC
- SapI -> mutG, oligo2
- BsaI -> mutT
- EciI -> mutA
- Restriction Digest:
- 1 µL Enzyme
- 5 µL CutSmart
- 25 µL mutC dsDNA
- 19 µL nuclease-free H2O
- Incubation: 37 °C, 1 h
- Inactivation: 20 min., 65 °C
- Native DNA PAGE standard protocol
2017-09-18 - 2017-09-24
getting positive clones for further work
Investigators: Yannic Kerkhoff
Superior experiment: heatshock transformation of the EutS-part from CU Boulder
Procedure:
- standard heatshock protocol
- used own produced chemo competend DH5alpha
- streaked out on LB-plates with Amp
getting positive clones for further work
Investigators: Yannic Kerkhoff
Superior experiment: heatshock transformation of the FusionRedTag-part from CU Boulder
Procedure:
- standard heatshock protocol
- used own produced chemo competend DH5alpha
- streaked out on LB-plates with Kan
getting positive clones for further work
Investigators: Yannic Kerkhoff
Superior experiment: heatshock cotransformation of the EutS-part and FusionRedTag-part from CU Boulder
Procedure:
- standard heatshock protocol
- used own produced chemo competend DH5alpha
- streaked out on LB-plates with AmpKan
Agarose gel electrophoresis of digested mutA, mutT, mutG & mutC
Investigators: Camilla Maerz
Superior experiment: M.A.X restriction system
Procedure:
- Agarose gel electrophoresis (2%)
- 60 V, 40 min
Annealing, restriction digest and native PAGE of all samples
Investigators: Camilla Maerz
Superior experiment: M.A.X restriction system
Procedure:
- Aqua annealing standard protocol (final concentration: 0.5 µM)
- mutA: 17jf & 17jg
- mutT: 17jj & 17jk
- mutG: 17jh & 17ji
- mutC: 17jl & 17jm
- oligo2: olgio2_sense & oligo2_antisense
- final volume of each sample: 2x50 µL
- Restricted samples:
- MnlI -> mutC
- SapI -> mutG, oligo2
- BsaI -> mutT
- EciI -> mutA
- Restriction Digest:
- 1 µL Enzyme
- 5 µL CutSmart
- 25 µL mutC dsDNA
- 19 µL nuclease-free H2O
- Incubation: 37 °C, 1 h
- Inactivation: 20 min., 65 °C
- Native DNA PAGE standard protocol
Combination of two biobricks to get the part BBa_K2201200 in a tetracycline low copy plasmid
Investigators: Yannic Kerkhoff
Superior experiment: Biobrick Assembly of BBa_K2201200 in psB3T5
Procedure:
- Standard BioBrick Assembly protocol
- Backbone: pSB3T5, 2,0µL
- upstream: BBa_K2201200, 3,6µL
- Ligation time was 12 h at 8°C
- cultivation
glycerine stocks of K2201200, K2201220, K2201221, K2201320, K2201321
Investigators: Yannic Kerkhoff
Superior experiment: glycerine stocks of K2201200, K2201220, K2201221, K2201320, K2201321
Procedure:
Screening for positive transformations containing the Biobrick
Investigators: Yannic Kerkhoff
Superior experiment: GoTaq PCR of 30 clones of K2201200 in pSB3T5
Procedure:
- standard GoTaq-protocol
- template: colonie pcr clones of K2201200 in pSB3T5
- primer fwd: Präfix_fwd
- primer rev: Suffix_rev
- annealing temperature: 56°C
- extension time: 40 seconds
- positive clones 1, 3, 6
- Preculture
Colony PCR of pSB3K5_mRFP_link_ccdB and pSB1C3_PtNTT2(31-575)-GFP
Investigators: Camilla Maerz
Superior experiment: Construction of pSB1C3-PlacUV5_PtNTT2
Procedure:
- PCR
- Go Tag (Promega) protocol
- Primer: VR, VF2
- Danaturation: 56 °C
- Elongation: 90 s & 180 s
- Agarose gel electrophoresis (1%)
- 100 V, 30 min
2017-09-25 - 2017-10-01
getting positive clones for further work
Investigators: Yannic Kerkhoff
Superior experiment: heatshock cotransformation of K2201200 in pSB3T5 and K2201321
Procedure:
- standard heatshock protocol
- used own produced chemo competend BL21(DE3)
- streaked out on LB-plates with TetCam
getting plasmids containing the Biobrick for further experiments
Investigators: Yannic Kerkhoff
Superior experiment: plasmid isolation of preculture of BBa_K2201207
Procedure:
- standard protocol
- 75,6 ng/µL
- Biobrick Assembly
Aqua annealing of mutA-1, mutG-1, mutC-1, mutG-1 & oligo1
Investigators: Camilla Maerz
Superior experiment: M.A.X restriction system
Procedure:
- Aqua annealing standard protocol (final concentration: 0.5 µM)
- mutA-1: 17iv & 17iw
- mutT-1: 17jb & 17jc
- mutG-1: 17ix & 17iy
- mutC-1: 17jd & 17jc
- oligo1: olgio1_sense & oligo1_antisense
- final volume: 50 µL
getting positive clones for further work
Investigators: Yannic Kerkhoff
Superior experiment: heatshock cotransformation of K2201240 and K2201207
Procedure:
- standard heatshock protocol
- used own produced chemo competend BL21(DE3)
- streaked out on LB-plates with AmpCam
getting positive clones for further work
Investigators: Yannic Kerkhoff
Superior experiment: heatshock cotransformation of K2201241 and K2201207
Procedure:
- standard heatshock protocol
- used own produced chemo competend BL21(DE3)
- streaked out on LB-plates with AmpCam
- Q5-PRC + Gel + cleanUp
2017-10-02 - 2017-10-08
UBP-PCR with TiTaq
Investigators: Camilla Maerz
Superior experiment: PCR with UBP
Procedure:
- PCR
- PCR-UBP standard protocol (TiTaq)
- samples:
- Ligation of: mutA, mutT, mutG, mutC and oligo 2 (1 ng/µL)
- Primer: 17vt & 17vu
- Primer annealing: 56 °C
- Elongation: 20 s
- PCR and DNA purification kit (Macherey-Nagel)
- Nucleo spin PCR and Monarch® PCR & DNA Clean-up protocol
- Restriction Digest
- 1 µL Enzyme
- 5 µL CutSmart
- 25 µL mutC dsDNA
- 19 µL nuclease-free H2O
- Incubation: 37 °C, 1 h
- Inactivation: 20 min., 65 °C
- Restricted samples:
- MnlI -> mutC
- SapI -> mutG
- BsaI -> mutT
- EciI -> mutA
- EciI, BsaI, SapI, MnlI -> oligo2
- Variation: oligo2 was digested with 0.5 µL of each enzyme
- Agarose gel electrophoresis (2 %)
- 100 V, 20 min
Generating the library
Investigators: Laura Schlueter
Superior experiment: aaRS plasmid
Procedure:
- Gibson Assembly Master Mix + dsDNA (3.5 µl) + library Plasmid (0.5 µL) with the opotical controle (25x)
- transformation in DH5a via heatshock (25 x)
Kanymycin with amber stop codon
Investigators: Laura Schlueter
Superior experiment: positiv selection plasmid
Procedure:
- amplification (~850 bp) of the kanamycin with primers 17jo, 17jp (no second MET codon) 71°C
- amplification (~850 bp) of the kanamycin with primers 17jr, 17jp (two amber stop codon) 71°C
Generating the library
Investigators: Laura Schlueter
Superior experiment: aaRS plasmid
Procedure:
- Gibson Assembly Master Mix + dsDNA (3.5 µl) + library Plasmid (0.5 µL) with the opotical controle (25x)
- transformation in DH5a via heatshock (25 x)
UBP-PCR with GoTaq G2 and TiTaq
Investigators: Camilla Maerz
Superior experiment: PCR with UBP
Procedure:
- PCR with TiTaq:
- PCR-UBP standard protocol (TiTaq)
- samples:
- Ligation of: mutA, mutT, mutG, mutC and oligo 2 (5 ng/µL)
- Primer: 17 vt & 17 vu
- Primer annealing: 56 °C
- Elongation: 20 s
- PCR with TiTaq:
- PCR-UBP standard protocol (TiTaq)
- samples:
- Ligation of: mutA, mutT, mutG and mutC (5 ng/µL)
- Primer: VR & VF2
- Primer annealing: 56 °C
- Elongation: 20 s
- PCR with GoTaq:
- PCR-UBP standard protocol (GoTaq)
- samples:
- Ligation of: mutA, mutT, mutG, mutC and oligo2 (5 ng/µL)
- Primer: 17 vt & 17 vu
- Primer annealing: 56 °C
- Elongation: 20 s
cloning of the aaRS in pSB3C5 (low copy)
Investigators: Laura Schlueter
Superior experiment: aaRS growth experiment
Procedure:
- restriction of pSB3C5 and aaRS+tRNA (NPA, AcF, Prk) with EcoRI and PstI following the restriction protocol
- extraction from the gel (Macherey-Nagel Kit)
- ligation of pSB3C5 and aaRS+tRNA (NPA, AcF, Prk) with T4 Ligase (NEB)
- Trafo via heatshock
sceening of the kanamycin (03.10.2017)
Investigators: Laura Schlueter
Superior experiment: positiv selection plasmid
Procedure:
- Colony PCR (GoTaq, Primer VF2, VR, 56.5 °C)
Restriction digest of ligated oligo2
Investigators: Camilla Maerz
Superior experiment: PCR with UBP
Procedure:
- Restriction Digest
- 5 µL CutSmart
- 30 µL ligated oligo2 DNA
- 2 µL EcoRV
- filled up to final volume 50 µL with ddH20
- Incubation: 120 min, 37 °C
- Inactivation: 20 min, 65 °C
Gradient PCR of mutT
Investigators: Camilla Maerz
Superior experiment: PCR with UBP
Procedure:
- PCR
- PCR-UBP standard protocol (GoTaq G2)
- sample: mutT (5 ng/µL)
- Primer: 17 vt & 17 vu
- Primer annealing: 50 - 56 °C
- Elongation: 20 s
- Agarose gel electrophoresis (2%)
- 100 V, 30 min
PCR of mutA, mutT, mutG & mutC with UBP and oligo2 without UBP
Investigators: Camilla Maerz
Superior experiment: PCR with UBP
Procedure:
- PCR with TiTaq:
- PCR-UBP standard protocol (TiTaq)
- samples:
- Ligation of: mutA, mutT, mutG and mutC (5 ng/µL), oligo2 (25 ng/µL)
- Primer: VR & VF2
- Primer annealing: 56 °C
- Elongation: 20 s
- Variation:
- oligo2 without UBP, instead 1 µL H20 was added
- 0.5 µL isoG & 0.5 µL isoCMe were added to mutA, mutT, mutG and mutC
Generating the library
Investigators: Laura Schlueter
Superior experiment: aaRS Plasmid
Procedure:
- plasmid isolation of the Tyr-RS library-mix
- we stamped out the not randomized, negative, clones, easy to identify by the red colour because of the mRFP
- washed from the cultivation plates with LB-media
- restriction of the Library with EcoRI and PstI
- proof of the restriction via agarose-gelelectrophoresis, 1 %
- positve result: bands on ~2000 bp (backbone) and ~1000 bp (Tyr-RS with NNK)
Generating the library
Investigators: Laura Schlueter
Superior experiment: aaRS plasmid
Procedure:
- Gibson Assembly Master Mix + dsDNA (3.5 µl) + library Plasmid (0.5 µL) with the opotical controle (25x)
- transformation in DH5a via heatshock (25 x)
2017-10-09 - 2017-10-15
PCR with switched primers: VF2 & 17vu, VR & 17vt
Investigators: Camilla Maerz
Superior experiment: PCR with UBP
Procedure:
- PCR with TiTaq:
- PCR-UBP standard protocol (TiTaq)
- samples:
- Ligation of: mutA, mutT, mutG and mutC (5 ng/µL), oligo2 (25 ng/µL)
- Primer: VF2 & 17vu, VR & 17vt
- Primer annealing: 56 °C
- Elongation: 20 s
- PCR with GoTaq:
- PCR-UBP standard protocol (GoTaq)
- samples:
- Ligation of: mutA, mutT, mutG and mutC (5 ng/µL), oligo2 (25 ng/µL)
- Primer: VF2 & 17vu, VR & 17vt
- Primer annealing: 56 °C
- Elongation: 20 s
- Agarose gel electrophoresis (2%)
- 100 V, 30 min
Generating the library
Investigators: Laura Schlueter
Superior experiment: aaRS plasmid
Procedure:
- Gibson Assembly Master Mix + dsDNA (3.5 µl) + library Plasmid (0.5 µL) with the opotical controle (25x)
- transformation in DH5a via heatshock (25 x)
PCR with higher DNA template concentrations and gradient PCR of mutT
Investigators: Camilla Maerz
Superior experiment: PCR with UBP
Procedure:
- PCR with TiTaq:
- PCR-UBP standard protocol (TiTaq)
- samples:
- Ligation of: mutT (5 ng/µL) and oligo2 (50 ng/µL)
- Primer: VF2 & 17vu
- Primer annealing: 56 °C
- Elongation: 20 s
- PCR and DNA purification kit (Macherey-Nagel)
- Nucleo spin PCR and Monarch® PCR & DNA Clean-up protocol
- Restriction Digest
- 0.5 µL Enzyme
- 2.3 µL CutSmart
- 20 µL dsDNA
- Incubation: 37 °C, 1 h
- Inactivation: 20 min., 65 °C
- Restricted samples:
- BsaI -> mutT
- EciI, BsaI, SapI, MnlI -> oligo2
- Variation: oligo2 was digested with 0.5 µL of each enzyme
- Agarose gel electrophoresis (2 %)
- 100 V, 20 min
- Gradient PCR with TiTaq:
- PCR-UBP standard protocol (TiTaq)
- samples:
- Ligation of: mutT (5 ng/µL)
- Primer: VF2 & 17vu
- Primer annealing: 50 - 58 °C
- Elongation: 20 s
- Agarose gel electrophoresis (2%)
- 100 V, 30 min
PCR of mutA, mutT, mutG, mutC and oligo2
Investigators: Camilla Maerz
Superior experiment: PCR with UBP
Procedure:
- PCR with GoTaq:
- PCR-UBP standard protocol (GoTaq)
- samples:
- Ligation of: mutA, mutT, mutG, mutC (5 ng/µL) and oligo2 (25 ng/µL)
- Primer: VF2 & 17vu
- Primer annealing: 56 °C
- Elongation: 20 s
- PCR and DNA purification kit (Macherey-Nagel)
- Nucleo spin PCR and Monarch® PCR & DNA Clean-up protocol
- Restriction Digest
- 1 µL Enzyme
- 2.3 µL CutSmart
- 20 µL dsDNA
- Incubation: 37 °C, 12 h
- Inactivation: 20 min., 65 °C
- Restricted samples:
- MnlI -> mutC
- SapI -> mutG
- BsaI -> mutT
- EciI -> mutA
- EciI, BsaI, SapI, MnlI -> oligo2
- Agarose gel electrophoresis (2 %)
- 100 V, 30 min
positive selection with the library
Investigators: Laura Schlueter
Superior experiment: pos selection
Procedure:
- using cultivation plates with kan (0.5 mM), Cm (0.25 mM), Tet (0.5 mM)
- no growth
Variation of restriction digest
Investigators: Camilla Maerz
Superior experiment: PCR with UBP
Procedure:
- Restriction Digest
- 3 µL Enzyme
- 2.5 µL CutSmart
- 20 µL dsDNA
- Incubation: 37 °C, 1 h
- Inactivation: 20 min., 65 °C
- Restricted samples:
- MnlI -> mutC
- SapI -> mutG
- BsaI -> mutT
- EciI -> mutA
- EciI, BsaI, SapI, MnlI -> oligo2
- Agarose gel electrophoresis (2 %)
- 100 V, 30 min
positive selection with the library
Investigators: Laura Schlueter
Superior experiment: pos selection
Procedure:
- Transformation (heatshock) of the library plasmid mix in competent cells containing the positive selction plasmid
- using cultivation plates with kan (0.3 mM), Cm (0.25 mM), Tet (0.5 mM)
- no growth
positive selection with the library
Investigators: Laura Schlueter
Superior experiment: pos selection
Procedure:
- Transformation (heatshock) of the library plasmid mix in competent cells containing the positive selction plasmid
- extended regeneration time: adding 0,5 mM IPTG after 1 h in SOC media
- using cultivation plates with kan (0.3 mM), Cm (0.25 mM), Tet (0.5 mM), IPTG (0,5 mM)
- no growth
growth experiments with the aaRS
Investigators: Laura Schlueter
Superior experiment: pos selection
Procedure:
- comparing AcF-TAG, AcF-Leu, Cou-AS, Prk, NPA (on low and high copy plasmid) pSB1C3, pSB3T5
- cultivation:
- LB media, Cm (0.25 mM) / Tet (0.5 mM) and 1 mM of the ncAA
- 12 microtiter well plate, 600 rpm, 37°C
2017-10-16 - 2017-10-22
PCR with TiTaq and Q5-polymerase
Investigators: Camilla Maerz
Superior experiment: PCR with UBP
Procedure:
- PCR with TiTaq:
- PCR-UBP standard protocol (TiTaq)
- samples:
- Ligation of: mutA, mutT, mutG & mutC (5 ng/µL) and oligo2 (50 ng/µL)
- Primer: VF2 & 17vu
- Primer annealing: 56 °C
- Elongation: 20 s
- PCR and DNA purification kit (Macherey-Nagel)
- Nucleo spin PCR and Monarch® PCR & DNA Clean-up protocol
- PCR with Q5 High-Fidelity Polymerase-polymerase (per reaction, 25 µL):
- 5 µL Reaction buffer
- 1.25 µL dNTPs (2 mM)
- 1.25 µL 3'-Primer (10 mM)
- 1.25 µL 5'-Primer (10 mM)
- 1 µL DNA template
- 0.25 µL Q5 Hifi-Polymerase
- 14 µL H20
- mutA, mutT, mutG, mutC: + 1 µL H20
- oligo2: + 0.5 µL isoCme, + 0.5 µL isoG
- Primer: VF2 & 17vu
- Primer annealing: 56 °C
- Elongation: 10 s
- PCR and DNA purification kit (Macherey-Nagel)
- Nucleo spin PCR and Monarch® PCR & DNA Clean-up protocol
- Restriction Digest
- 1 µL Enzyme
- 20 µL dsDNA
- 2.3 µL CutSmart Buffer
- Incubation: 37 °C, 15 h (BsaI & MnlI), 2 h (SapI & EciI)
- Inactivation: 65 °C, 20 min
- Restricted samples:
- MnlI -> mutC
- SapI -> mutG
- BsaI -> mutT
- EciI -> mutA
- EciI, BsaI, SapI, MnlI -> oligo2
PCR with different polymerases
Investigators: Camilla Maerz
Superior experiment: PCR with UBP
Procedure:
- samples:
- Ligation of: mutA, mutT, mutG & mutC (5 ng/µL) and oligo2 (50 ng/µL)
- PCR with Allin Hifi DNA Polymerase (per reaction, 25 µL):
- 5 µL ReactionBuffer
- 0.25 µL Hifi DNA Polymerase
- 2.5 µL 3'-Primer (10 mM)
- 2.5 µL 5'-Primer (10 mM)
- 1 µL DNA template
- 12.75 µL H20
- mutA, mutT, mutG, mutC: + 1 µL H20
- oligo2: + 0.5 µL isoCme, + 0.5 µL isoG
- Primer: VF2 & 17vu
- Primer annealing: 56 °C
- Elongation: 10 s
- PCR and DNA purification kit (Macherey-Nagel)
- Nucleo spin PCR and Monarch® PCR & DNA Clean-up protocol
- PCR with innuDRY Standard PCR MasterMix (per reaction, 20 µL):
- 10 µL innduDRY Mastermix
- 2 µL 3'-Primer (10 mM)
- 2 µL 5'-Primer (10 mM)
- 1 µL DNA template
- 5 µL H20
- mutA, mutT, mutG, mutC: + 1 µL H20
- oligo2: + 0.5 µL isoCme, + 0.5 µL isoG
- Primer: VF2 & 17vu
- Primer annealing: 54 °C
- Elongation: 30 s
- PCR and DNA purification kit (Macherey-Nagel)
- Nucleo spin PCR and Monarch® PCR & DNA Clean-up protocol
- PCR with BioMaster-HS Taq PCR Color (per reaction, 25 µL):
- 12.5 µL Mastermix
- 0.7 µL 3'-Primer (10 mM)
- 0.7 µL 5'-Primer (10 mM)
- 1 µL DNA template
- 10.1 µL H20
- mutA, mutT, mutG, mutC: + 1 µL H20
- oligo2: + 0.5 µL isoCme, + 0.5 µL isoG
- Primer: VF2 & 17vu
- Primer annealing: 51 °C
- Elongation: 20 s
- PCR and DNA purification kit (Macherey-Nagel)
- Nucleo spin PCR and Monarch® PCR & DNA Clean-up protocol
- PCR with Fire Polymerase (per reaction, 20 µL):
- 4 µL Mastermix
- 0.5 µL 3'-Primer (10 mM)
- 0.5 µL 5'-Primer (10 mM)
- 1 µL DNA template
- 14 µL H20
- mutA, mutT, mutG, mutC: + 1 µL H20
- oligo2: + 0.5 µL isoCme, + 0.5 µL isoG
- Primer: VF2 & 17vu
- Primer annealing: 56 °C
- Elongation: 20 s
- PCR and DNA purification kit (Macherey-Nagel)
- Nucleo spin PCR and Monarch® PCR & DNA Clean-up protocol
- PCR with Phusion polymerase (per reaction, 25 µL):
- 12.5 µL Mastermix
- 1.25 µL 3'-Primer (10 mM)
- 1.25 µL 5'-Primer (10 mM)
- 1 µL DNA template
- 9 µL H20
- mutA, mutT, mutG, mutC: + 1 µL H20
- oligo2: + 0.5 µL isoCme, + 0.5 µL isoG
- Primer: VF2 & 17vu
- Primer annealing: 56 °C
- Elongation: 20 s
- PCR and DNA purification kit (Macherey-Nagel)
- Nucleo spin PCR and Monarch® PCR & DNA Clean-up protocol
- Restriction Digest
- 1 µL Enzyme
- 20 µL dsDNA
- 2.3 µL CutSmart Buffer
- Incubation: 37 °C, 15 h (BsaI & MnlI), 2 h (SapI & EciI)
- Inactivation: 65 °C, 20 min
- Restricted samples:
- MnlI -> mutC
- SapI -> mutG
- BsaI -> mutT
- EciI -> mutA
- EciI, BsaI, SapI, MnlI -> oligo2
PCR with different polymerases
Investigators: Camilla Maerz
Superior experiment: PCR with UBP
Procedure:
- Agarose gel electrophoresis with all samples (17.10)
- Agarose gel electrophoresis (2%)
- 100 V, 40 min