Difference between revisions of "Team:Bielefeld-CeBiTec/Notebook/Labjournal"

(added first version of labjournal)
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</div>
 
</div>
 
</div>
 
</div>
 +
 +
 +
            <div class="labnotebox" style="margin-top: 50px;">
 +
<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>Aim of the 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>Aim of the 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>Aim of the experiment:</b> preculture of transformed BBa_K1416000</p>
 +
<p><b>Procedure:</b></p>
 +
<article>
 +
<ul>
 +
<li>  standard procedure</li>
 +
<ul>
 +
<li>  1,25֬ 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>Aim of the 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/֌</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>Aim of the experiment:</b> preculture of transformed BBa_J36848</p>
 +
<p><b>Procedure:</b></p>
 +
<article>
 +
<ul>
 +
<li>  standard procedure</li>
 +
<ul>
 +
<li>  1,25֬ 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>Aim of the 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/֌</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>Aim of the 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>Aim of the experiment:</b> preculture of transformed BBa_E0400</p>
 +
<p><b>Procedure:</b></p>
 +
<article>
 +
<ul>
 +
<li>  standard procedure</li>
 +
<ul>
 +
<li>  5,0֬ 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>Aim of the 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/֌</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>Aim of the 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>Aim of the experiment:</b> preculture of transformed BBa_K525998</p>
 +
<p><b>Procedure:</b></p>
 +
<article>
 +
<ul>
 +
<li>  standard procedure</li>
 +
<ul>
 +
<li>  1,25֬ 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>Aim of the 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/֌</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 composite Part BBa_P1:GLS1 </h3>
 +
</div>
 +
<div class="labnote-content">
 +
<p><b>Investigators:</b> Yannic Kerkhoff</p>
 +
<p><b>Aim of the 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> To get and purified the plasmid </h3>
 +
</div>
 +
<div class="labnote-content">
 +
<p><b>Investigators:</b> Christina Drake</p>
 +
<p><b>Aim of the 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>Aim of the 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>Aim of the 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>Aim of the 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>Aim of the 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>Aim of the 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>Aim of the 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>Aim of the 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: 58у</li>
 +
<li>  extension time: 50 seconds</li>
 +
</ul>
 +
<li>  standard PCR-cleanUp protocol</li>
 +
<ul>
 +
<li>  125,8 ng/֌</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>Aim of the 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: 61у</li>
 +
<li>  extension time: 30 seconds</li>
 +
</ul>
 +
<li>  standard PCR-cleanUp protocol</li>
 +
<ul>
 +
<li>  127,0 ng/֌</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>Aim of the 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: 59у</li>
 +
<li>  extension time: 120 seconds</li>
 +
</ul>
 +
<li>  standard PCR-cleanUp protocol</li>
 +
<ul>
 +
<li>  258,4 ng/֌</li>
 +
</ul>
 +
</article>
 +
</div>
 +
</div>
 +
<div class="labnotebox">
 +
<div class="labnote-title">
 +
<h3> Assemble the fragments to get the composite Part BBa_P2:GLS2 </h3>
 +
</div>
 +
<div class="labnote-content">
 +
<p><b>Investigators:</b> Yannic Kerkhoff</p>
 +
<p><b>Aim of the 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 BBa_P2:GLS2 </h3>
 +
</div>
 +
<div class="labnote-content">
 +
<p><b>Investigators:</b> Yannic Kerkhoff</p>
 +
<p><b>Aim of the experiment:</b> GoTaq PCR of seven clones</p>
 +
<p><b>Procedure:</b></p>
 +
<article>
 +
<ul>
 +
<li>  standard GoTaq-protocol</li>
 +
<ul>
 +
<li>  template: colonie pcr clones of GA_P2:GLS2</li>
 +
<li>  primer fwd: Pr姩x_fwd</li>
 +
<li>  primer rev: Suffix_rev</li>
 +
<li>  annealing temperature: 56у</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>Aim of the experiment:</b> preculture of the positive clone 2 of BBa_P2:GLS2</p>
 +
<p><b>Procedure:</b></p>
 +
<article>
 +
<ul>
 +
<li>  standard procedure</li>
 +
<ul>
 +
<li>  1,25֬ 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>Aim of the experiment:</b> plasmid isolation of preculture of BBa_P2:GLS2</p>
 +
<p><b>Procedure:</b></p>
 +
<article>
 +
<ul>
 +
<li>  standard protocol</li>
 +
<ul>
 +
<li>  208,8 ng/֌</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>Aim of the 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> PCR clean up of gblock1, gblock2 and pK18mobsacB-backbone </h3>
 +
</div>
 +
<div class="labnote-content">
 +
<p><b>Investigators:</b> Camilla Maerz</p>
 +
<p><b>Aim of the experiment:</b> Construction of pK18mobsacB_codA_del</p>
 +
<p><b>Procedure:</b></p>
 +
<article>
 +
<ul>
 +
<li> PCR and DNA purification kit</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>
 +
</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>Aim of the 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> Isolate T7RNAP from the chromosome of KRX-e.coli </h3>
 +
</div>
 +
<div class="labnote-content">
 +
<p><b>Investigators:</b> Christina Drake</p>
 +
<p><b>Aim of the 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>Aim of the 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>Aim of the 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 у</li>
 +
<li> Elongation: 140 s</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> t </h3>
 +
</div>
 +
<div class="labnote-content">
 +
<p><b>Investigators:</b> Christina Drake</p>
 +
<p><b>Aim of the experiment:</b> Plasmidisolation of Barnase and colonies 1-5 T7RNAP</p>
 +
<p><b>Procedure:</b></p>
 +
<article>
 +
<li> To get and purified the plasmid</li>
 +
<ul>
 +
<li>  protocol from Macherley-Nagel: Plasmid DNA purification, protocol 5: NucleoSpin</li>
 +
<li>  concentration measurement by nanoprop</li>
 +
</ul>
 +
<li> >03.07.2017</li>
 +
<li> tRNA</li>
 +
<li> PCR of barnase</li>
 +
<li> CDR</li>
 +
<li> Check the correct barnasegen</li>
 +
<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>Aim of the 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>Aim of the 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>Aim of the 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> Preculture of colonies with T7-Promotor </h3>
 +
</div>
 +
<div class="labnote-content">
 +
<p><b>Investigators:</b> Maximilian Edich</p>
 +
<p><b>Aim of the 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>Aim of the 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>Aim of the 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>Aim of the 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>Aim of the 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>Aim of the experiment:</b> Interlab study</p>
 +
<p><b>Procedure:</b></p>
 +
<article>
 +
<ul>
 +
<li> dilute test devives (distribution plate 6 & 7) in 10 ֌ ddH20</li>
 +
<li> Transformation</li>
 +
<ul>
 +
<li> 1 ֌ 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>Aim of the 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>Aim of the 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>Aim of the 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>Aim of the 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> Preparation of over-night culture </h3>
 +
</div>
 +
<div class="labnote-content">
 +
<p><b>Investigators:</b> Camilla Maerz</p>
 +
<p><b>Aim of the 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 (37у, 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> 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>Aim of the 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>Aim of the 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>
 +
<li> >31.07.2017</li>
 +
<li> tRNA</li>
 +
<li> Aquacloning of tRNA and backbone</li>
 +
<li> CDR</li>
 +
<li> construct a biobrick</li>
 +
<ul>
 +
<li> transformation via heatschock</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>Aim of the 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 ֌    Ori 1.0: 1,73 ֌</li>
 +
<li> Backbone: 0,32 ֌    Ori 1.1: 4,68 ֌</li>
 +
<li> Backbone: 0,90 ֌    Ori 3.0: 4,10 ֌</li>
 +
</ul>
 +
<li> Trafo: Hitzeschock</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>Aim of the 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>Aim of the 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, 65у</li>
 +
<li> gelelectrophoresis, 1% Agarose</li>
 +
<li> band at 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/֌</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>Aim of the 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, 65у</li>
 +
<li> Primer jb, ht for fragment 4, 65у</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/֌  (overlapp(aaRS)_pSB1C3_overlapp(ori))</li>
 +
<li> fragment 4: 19,1 ng/֌  (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>Aim of the 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>Aim of the 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 : stron bands at around 1200 bp</li>
 +
</ul>
 +
<li> because of the the two positive colony PCRs (vf-vr, hq-jk) for these probes, the 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>Aim of the experiment:</b> aaRS plasmid</p>
 +
<p><b>Procedure:</b></p>
 +
<article>
 +
<ul>
 +
<li> Macherey-Nagel purification kit</li>
 +
<ul>
 +
<li> pRS31: 324,1 ng/֌</li>
 +
<li> pRS32: 231,8 ng/֌</li>
 +
<li> pRS39: 225,5 ng/֌</li>
 +
</ul>
 +
<li> send to the sequencing</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> 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>Aim of the 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/֌</li>
 +
<li> pRS3,12: 68,3 ng/֌</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>Aim of the 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 ֬ template</li>
 +
<ul>
 +
<li> pSB1C3 fragment (2):      18,5 ng/֌  around  350 bp  2,2 ֌</li>
 +
<li> pSB1C3 fragment (4):      19,1 ng/֌  around 1300 bp  0,8 ֌</li>
 +
<li> oK1.1 (pMB1 from pSB6A1):  5,8 ng/֌  around 1300 bp  1,8 ֌</li>
 +
<li> Tyr-aaRS:                  68,5 ng/֌  around 1000 bp  0,2 ֌</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>Aim of the 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 ֌ template</li>
 +
<ul>
 +
<li> pSB1C3(nur ori): 22,2  ng/֌  around 2100 bp  1,57 ֌</li>
 +
<li> ok1.0:            5,80 ng/֌  around 1258 bp  3,43 ֌</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>Aim of the 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>Aim of the 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>Aim of the experiment:</b> Gibbsonassembly of barnase and backbone</p>
 +
<p><b>Procedure:</b></p>
 +
<article>
 +
<ul>
 +
<li> transformation via heatschock</li>
 +
</ul>
 +
<li> >03.08.2017</li>
 +
<li> tRNA</li>
 +
<li> Overnightculture of tRNA-psB1C3 and barnase-psB1C3</li>
 +
<li> CDR</li>
 +
<li> Cellgrowth with the 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> Preculture of 2B6, 2B7 and A1 </h3>
 +
</div>
 +
<div class="labnote-content">
 +
<p><b>Investigators:</b> Maximilian Edich</p>
 +
<p><b>Aim of the 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> Plasmidisolation </h3>
 +
</div>
 +
<div class="labnote-content">
 +
<p><b>Investigators:</b> Maximilian Edich</p>
 +
<p><b>Aim of the 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>Aim of the 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>Aim of the 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>Aim of the experiment:</b> aaRS plasmid</p>
 +
<p><b>Procedure:</b></p>
 +
<article>
 +
<ul>
 +
<li> PCR Q5 Polymerase (no Master Mix)</li>
 +
<ul>
 +
<li> for 50 ֌ reaction</li>
 +
<ul>
 +
<li> 10  ֌ Reaction Buffer</li>
 +
<li>  5  ֌ 2mM dNTPs</li>
 +
<li>  1  ֌ Template</li>
 +
<li>  0,5 ֌ High Fidelity DNA Polymerase</li>
 +
<li> 10,0 ֌ High GC Enhancer</li>
 +
<li> 18,5 ֌ ddest H2O</li>
 +
<li>  5,0 ֌ Primer</li>
 +
<ul>
 +
<li> pSB1C3 (from RuBisCo, ncAA team): ht, jk  65у</li>
 +
<li> aaRS (from Heidelberg) : hq, jk 65у (mistake! should be 58у, redone identically with 58у)</li>
 +
</ul>
 +
<li> digestion of the PCR products with Dpn1: 5 ֌ CutSmart Reaktionsbuffer and 1 ֌ Dpn1 per 50 ֌ PCR product, on 37у 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>Aim of the 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>Aim of the 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>
 +
<li> >08.08.2017</li>
 +
<li> tRNA</li>
 +
<li> PCR of T7RNA-Polymerase</li>
 +
<li> CDR</li>
 +
<li> construct a biobrick for the selectionplasmid</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>
 +
<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>Aim of the 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  65у</li>
 +
<li> oK1:  primer jl, jc  65у</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>Aim of the 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/֌</li>
 +
<li> pRS32:  9,5 ng/֌</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 ֌ of oK1.1: around 2480 bp  9,5 ng/֌</li>
 +
<li> 0,9 ֌ of pRS32: around 1250 bp 22,5 ng/֌</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>Aim of the 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>Aim of the 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: 63у</li>
 +
<li>  extension time: 90 seconds</li>
 +
</ul>
 +
<li>  standard PCR-cleanUp protocol</li>
 +
<ul>
 +
<li>  80,6 ng/֌</li>
 +
</ul>
 +
</article>
 +
</div>
 +
</div>
 +
<div class="labnotebox">
 +
<div class="labnote-title">
 +
<h3> Assemble the fragments to get the composite Part BBa_P7:NPA-RS </h3>
 +
</div>
 +
<div class="labnote-content">
 +
<p><b>Investigators:</b> Yannic Kerkhoff</p>
 +
<p><b>Aim of the 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>Aim of the 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>Aim of the 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: 58у</li>
 +
<li>  extension time: 50 seconds</li>
 +
</ul>
 +
<li>  standard PCR-cleanUp protocol</li>
 +
<ul>
 +
<li>  15,9 ng/֌</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>Aim of the 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: 61у</li>
 +
<li>  extension time: 30 seconds</li>
 +
</ul>
 +
<li>  standard PCR-cleanUp protocol</li>
 +
<ul>
 +
<li>  17,9 ng/֌</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>Aim of the 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>Aim of the 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> Primer annealing preparation </h3>
 +
</div>
 +
<div class="labnote-content">
 +
<p><b>Investigators:</b> Camilla Maerz</p>
 +
<p><b>Aim of the 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 ֍, 10 ֍, 5 ֍, 2.5 ֍, 0.5 ֍</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 BBa_P2:GLS2 </h3>
 +
</div>
 +
<div class="labnote-content">
 +
<p><b>Investigators:</b> Yannic Kerkhoff</p>
 +
<p><b>Aim of the experiment:</b> GoTaq PCR of 4 clones</p>
 +
<p><b>Procedure:</b></p>
 +
<article>
 +
<ul>
 +
<li>  standard GoTaq-protocol</li>
 +
<ul>
 +
<li>  template: colonie pcr clones of GA_P1:GLS1</li>
 +
<li>  primer fwd: Pr姩x_fwd</li>
 +
<li>  primer rev: Suffix_rev</li>
 +
<li>  annealing temperature: 56у</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> Primer annealing of 17jh & 17ji </h3>
 +
</div>
 +
<div class="labnote-content">
 +
<p><b>Investigators:</b> Camilla Maerz</p>
 +
<p><b>Aim of the 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 ֌)</li>
 +
<li> Thermal profile (thermocycler)</li>
 +
<ul>
 +
<li> 95 у, 2 min</li>
 +
<li> Cool to 25 у over 45 min</li>
 +
<li> Cool to 4 у for temporary storage</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>Aim of the 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 ֍</li>
 +
<li> HEPES annealing standard protocol</li>
 +
<li> Aqua annealing standard protocol</li>
 +
<ul>
 +
<li> Final volume: 20 ֌</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> Native PAGE of annealed primers (17jl & 17jm) </h3>
 +
</div>
 +
<div class="labnote-content">
 +
<p><b>Investigators:</b> Camilla Maerz</p>
 +
<p><b>Aim of the 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 ֍, 10 ֍, 5 ֍, 2.5 ֍, 1 ֍, 0.5 ֍</li>
 +
<li> Aqua annealing (17jl & 17jm): 100 ֍, 10 ֍, 5 ֍, 2.5 ֍, 1 ֍, 0.5 ֍</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>Aim of the 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 ֍, 10 ֍, 5 ֍, 2.5 ֍, 1 ֍, 0.5 ֍</li>
 +
<li> Aqua annealing (17jl & 17jm): 100 ֍, 10 ֍, 5 ֍, 2.5 ֍, 1 ֍, 0.5 ֍</li>
 +
</ul>
 +
<li> Variation: 100 V, 1 h</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>Aim of the 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 ֍, 10 ֍, 5 ֍, 2.5 ֍, 1 ֍, 0.5 ֍</li>
 +
<li> Aqua annealing (17jl & 17jm): 100 ֍, 10 ֍, 5 ֍, 2.5 ֍, 1 ֍, 0.5 ֍</li>
 +
</ul>
 +
<li> Variation: 60 V, 2 h</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>Aim of the 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 ֍</li>
 +
<li> Aqua annealing (17jl & 17jm): 0.5 ֍</li>
 +
<li> Composite Buffer annealing (17jh & 17 jm): 0.5 ֍</li>
 +
<li> all Primers (ssDNA): 1 ֍</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>Aim of the 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 ֍</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> Restriction digest of control annealings (mutC) </h3>
 +
</div>
 +
<div class="labnote-content">
 +
<p><b>Investigators:</b> Camilla Maerz</p>
 +
<p><b>Aim of the 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 ֍)</li>
 +
<ul>
 +
<li> 2 ֌ <i>Mnl</i>I</li>
 +
<li> 5 ֌ CutSmart</li>
 +
<li> 25 ֌ mutC dsDNA</li>
 +
<li> 18 ֌ nuclease-free H2O</li>
 +
<li> Incubation: 37 у, 1 h</li>
 +
<li> Inactivation: 20 min., 65 у</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> Restriction digest of aqua & HEPES annealing </h3>
 +
</div>
 +
<div class="labnote-content">
 +
<p><b>Investigators:</b> Camilla Maerz</p>
 +
<p><b>Aim of the 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 ֍)</li>
 +
<ul>
 +
<li> 2 ֌ <i>Mnl</i>I</li>
 +
<li> 5 ֌ CutSmart</li>
 +
<li> 25 ֌ mutC dsDNA</li>
 +
<li> 18 ֌ nuclease-free H2O</li>
 +
<li> Incubation: 37 у, 1 h</li>
 +
<li> Inactivation: 20 min., 65 у</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>Aim of the experiment:</b> M.A.X restriction system</p>
 +
<p><b>Procedure:</b></p>
 +
<article>
 +
<ul>
 +
<li> Aqua annealing standard protocol (final concentration: 0.5 ֍)</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 ֌</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>Aim of the 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 ֍)</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 ֌ Enzyme</li>
 +
<li> 5 ֌ CutSmart</li>
 +
<li> 25 ֌ mutC dsDNA</li>
 +
<li> 19 ֌ nuclease-free H2O</li>
 +
<li> Incubation: 37 у, 1 h</li>
 +
<li> Inactivation: 20 min., 65 у</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> 2017-09-11  -  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>Aim of the experiment:</b> M.A.X restriction system</p>
 +
<p><b>Procedure:</b></p>
 +
<article>
 +
<ul>
 +
<li> Aqua annealing standard protocol (final concentration: 0.5 ֍)</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 ֌</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 ֌ Enzyme</li>
 +
<li> 5 ֌ CutSmart</li>
 +
<li> 25 ֌ mutC dsDNA</li>
 +
<li> 19 ֌ nuclease-free H2O</li>
 +
<li> Incubation: 37 у, 1 h</li>
 +
<li> Inactivation: 20 min., 65 у</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> Annealing, restriction digest and native PAGE of all samples </h3>
 +
</div>
 +
<div class="labnote-content">
 +
<p><b>Investigators:</b> Camilla Maerz</p>
 +
<p><b>Aim of the experiment:</b> M.A.X restriction system</p>
 +
<p><b>Procedure:</b></p>
 +
<article>
 +
<ul>
 +
<li> Aqua annealing standard protocol (final concentration: 0.5 ֍)</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 ֌</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 ֌ Enzyme</li>
 +
<li> 5 ֌ CutSmart</li>
 +
<li> 25 ֌ mutC dsDNA</li>
 +
<li> 19 ֌ nuclease-free H2O</li>
 +
<li> Incubation: 37 у, 1 h</li>
 +
<li> Inactivation: 20 min., 65 у</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> -Agarose gel electrophoresis (2%) </h3>
 +
</div>
 +
<div class="labnote-content">
 +
<p><b>Investigators:</b> Agarose, gel, electrophoresis, of, digested, mutA,, mutT,, mutG, &, mutC</p>
 +
<p><b>Aim of the experiment:</b> CMZ</p>
 +
<p><b>Procedure:</b></p>
 +
<article>
 +
<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>Aim of the experiment:</b> M.A.X restriction system</p>
 +
<p><b>Procedure:</b></p>
 +
<article>
 +
<ul>
 +
<li> Aqua annealing standard protocol (final concentration: 0.5 ֍)</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 ֌</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 ֌ Enzyme</li>
 +
<li> 5 ֌ CutSmart</li>
 +
<li> 25 ֌ mutC dsDNA</li>
 +
<li> 19 ֌ nuclease-free H2O</li>
 +
<li> Incubation: 37 у, 1 h</li>
 +
<li> Inactivation: 20 min., 65 у</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> 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>Aim of the 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 у</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> -Aqua annealing standard protocol (final concentration: 0.5 ֍) </h3>
 +
</div>
 +
<div class="labnote-content">
 +
<p><b>Investigators:</b> Aqua, annealing, of, mutA-1,, mutG-1,, mutC-1,, mutG-1, &, oligo1</p>
 +
<p><b>Aim of the experiment:</b> ??</p>
 +
<p><b>Procedure:</b></p>
 +
<article>
 +
<ul>
 +
<li> mutA-1: 17iv & 17iw</li>
 +
<ul>
 +
<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 ֌</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>Aim of the 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/֌)</li>
 +
</ul>
 +
<li> Primer: 17vt & 17vu</li>
 +
<li> Primer annealing: 56 у</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 ֌ Enzyme</li>
 +
<li> 5 ֌ CutSmart</li>
 +
<li> 25 ֌ mutC dsDNA</li>
 +
<li> 19 ֌ nuclease-free H2O</li>
 +
<li> Incubation: 37 у, 1 h</li>
 +
<li> Inactivation: 20 min., 65 у</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 ֌ 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> UBP-PCR with GoTaq G2 and TiTaq </h3>
 +
</div>
 +
<div class="labnote-content">
 +
<p><b>Investigators:</b> Camilla Maerz</p>
 +
<p><b>Aim of the 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/֌)</li>
 +
</ul>
 +
<li> Primer: 17 vt & 17 vu</li>
 +
<li> Primer annealing: 56 у</li>
 +
<li> Elongation: 20 s</li>
 +
</ul>
 +
<li> PCR with GoTaq:</li>
 +
<ul>
 +
<li> PCR-UBP standard protocol (TiTaq)</li>
 +
<li> samples:</li>
 +
<ul>
 +
<li> Ligation of: mutA, mutT, mutG and mutC (5 ng/֌)</li>
 +
</ul>
 +
<li> Primer: VR & VF2</li>
 +
<li> Primer annealing: 56 у</li>
 +
<li> Elongation: 20 s</li>
 +
</ul>
 +
<li> PCR with GoTaq:</li>
 +
<ul>
 +
<li> PCR-UBP standard protocol (TiTaq)</li>
 +
<li> samples:</li>
 +
<ul>
 +
<li> Ligation of: mutA, mutT, mutG, mutC and oligo2 (5 ng/֌)</li>
 +
</ul>
 +
<li> Primer: 17 vt & 17 vu</li>
 +
<li> Primer annealing: 56 у</li>
 +
<li> Elongation: 20 s</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>Aim of the 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 ֌ CutSmart</li>
 +
<li> 30 ֌ ligated oligo2 DNA</li>
 +
<li> 2 ֌ <i>Eco</i>RV</li>
 +
<li> filled up to final volume 50 ֌ with ddH20</li>
 +
<li> Incubation: 120 min, 37 у</li>
 +
<li> Inactivation: 20 min, 65 у</li>
 +
</ul>
 +
</article>
 +
</div>
 +
</div>
 +
            <div class="labnotebox">
 +
<div class="labnote-date">
 +
<h3> 2917-06-14  -  2917-06-20 </h3>
 +
</div>
 +
</div>
 +
<div class="labnotebox">
 +
<div class="labnote-title">
 +
<h3> Duplicate the KRX-e.coli </h3>
 +
</div>
 +
<div class="labnote-content">
 +
<p><b>Investigators:</b> Christina Drake, Denise Kerkhoff</p>
 +
<p><b>Aim of the experiment:</b> Plated out: KRX-e.coli as glycostocsolution</p>
 +
<p><b>Procedure:</b></p>
 +
<article>
 +
<ul>
 +
<li> plated out on kanamycin</li>
 +
<li> incubation at 37°C</li>
 +
</ul>
 +
</article>
 +
</div>
 +
</div>
 +
 +
  
 
</div>
 
</div>

Revision as of 13:13, 13 October 2017

Labjournal

2017-04-03 - 2017-04-09

getting positive clones for further work

Investigators: Yannic Kerkhoff

Aim of the experiment: electroporation transformation of BBa_K1416000

Procedure:

  • standard electroporation protocol
    • used electro competend DH5alpha from NEB
    • streaked out on LB-plates with Cam

getting positive clones for further work

Investigators: Yannic Kerkhoff

Aim of the 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

Aim of the experiment: preculture of transformed BBa_K1416000

Procedure:

  • standard procedure
    • 1,25֬ Cam in 5mL LB

2017-04-10 - 2017-04-16

getting plasmids containing the Biobrick for further experiments

Investigators: Yannic Kerkhoff

Aim of the experiment: plasmid isolation of preculture of BBa_K1416000

Procedure:

  • standard protocol
    • 517,9 ng/֌

multiplication of plasmids containing the Biobrick

Investigators: Yannic Kerkhoff

Aim of the experiment: preculture of transformed BBa_J36848

Procedure:

  • standard procedure
    • 1,25֬ Cam in 5mL LB

getting plasmids containing the Biobrick for further experiments

Investigators: Yannic Kerkhoff

Aim of the experiment: plasmid isolation of preculture of BBa_J36848

Procedure:

  • standard protocol
    • 231,8 ng/֌

2017-05-29 - 2017-06-04

getting positive clones for further work

Investigators: Yannic Kerkhoff

Aim of the 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

Aim of the experiment: preculture of transformed BBa_E0400

Procedure:

  • standard procedure
    • 5,0֬ Amp in 5mL LB

getting plasmids containing the Biobrick for further experiments

Investigators: Yannic Kerkhoff

Aim of the experiment: plasmid isolation of preculture of BBa_E0400

Procedure:

  • standard protocol
    • 88,4 ng/֌

getting positive clones for further work

Investigators: Yannic Kerkhoff

Aim of the 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

Aim of the experiment: preculture of transformed BBa_K525998

Procedure:

  • standard procedure
    • 1,25֬ Cam in 5mL LB

getting plasmids containing the Biobrick for further experiments

Investigators: Yannic Kerkhoff

Aim of the experiment: plasmid isolation of preculture of BBa_K525998

Procedure:

  • standard protocol
    • 140,9 ng/֌

2017-06-12 - 2017-06-18

Assemble the fragments to get the composite Part BBa_P1:GLS1

Investigators: Yannic Kerkhoff

Aim of the experiment: Gibson assembly of F1,F3,F5

Procedure:

To get and purified the plasmid

Investigators: Christina Drake

Aim of the 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

Aim of the experiment: Transformation via Distribution of the parts BBA_J04450, BBa_I746909, BBa_K80800 and BBa_psB1K5

Procedure:

Duplicate the part

Investigators: Christina Drake, Denise Kerkhoff

Aim of the experiment: Transformation of BBa_psB6A1

Procedure:

Cellgrowth with the plasmids

Investigators: Denise Kerkhoff, Christina Drake

Aim of the 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

Aim of the 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

Aim of the 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

Aim of the 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

Aim of the 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: 58у
    • extension time: 50 seconds
  • standard PCR-cleanUp protocol
    • 125,8 ng/֌

multiplication, verification and purification of the needed fragment F4: StrepLinker2

Investigators: Yannic Kerkhoff

Aim of the 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: 61у
    • extension time: 30 seconds
  • standard PCR-cleanUp protocol
    • 127,0 ng/֌

multiplication, verification and purification of the needed fragment F5: linearized pSB1C3

Investigators: Yannic Kerkhoff

Aim of the experiment: Q5-PCR, gel and PCR-cleanUp of F5

Procedure:

  • standard Q5-PCR protocol
    • template: K1416000
    • primer fwd: 17gj
    • primer rev: 17gl
    • annealing temperature: 59у
    • extension time: 120 seconds
  • standard PCR-cleanUp protocol
    • 258,4 ng/֌

Assemble the fragments to get the composite Part BBa_P2:GLS2

Investigators: Yannic Kerkhoff

Aim of the experiment: Gibson assembly of F2,F4,F5

Procedure:

Screening for positive transformations containing the Biobrick BBa_P2:GLS2

Investigators: Yannic Kerkhoff

Aim of the experiment: GoTaq PCR of seven clones

Procedure:

  • standard GoTaq-protocol
    • template: colonie pcr clones of GA_P2:GLS2
    • primer fwd: Pr姩x_fwd
    • primer rev: Suffix_rev
    • annealing temperature: 56у
    • extension time: 40 seconds
  • positive clone 2

multiplication of plasmids containing the Biobrick

Investigators: Yannic Kerkhoff

Aim of the experiment: preculture of the positive clone 2 of BBa_P2:GLS2

Procedure:

  • standard procedure
    • 1,25֬ Cam in 5mL LB

getting plasmids containing the Biobrick for further experiments

Investigators: Yannic Kerkhoff

Aim of the experiment: plasmid isolation of preculture of BBa_P2:GLS2

Procedure:

  • standard protocol
    • 208,8 ng/֌

2017-06-26 - 2017-07-02

Check the sequence

Investigators: Christina Drake

Aim of the experiment: Sequenzingorder barnase

Procedure:

  • concentration measurement by nanoprop: 177 ng/µl
  • result: sequence was not barnase

PCR clean up of gblock1, gblock2 and pK18mobsacB-backbone

Investigators: Camilla Maerz

Aim of the experiment: Construction of pK18mobsacB_codA_del

Procedure:

Plasmidisolation and gibson assembly of pSB1C3-PlacUV5_PtNTT2 c30

Investigators: Camilla Maerz

Aim of the experiment: Construction of pSB1C3-PlacUV5_PtNTT2

Procedure:

Isolate T7RNAP from the chromosome of KRX-e.coli

Investigators: Christina Drake

Aim of the 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

Aim of the 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

Aim of the experiment: Construction of pK18mobsacB_codA_del

Procedure:

  • Go Tag (Promega) protocol
    • Primer: VR, VF2
    • Primer annealing: 56 у
    • Elongation: 140 s

2017-07-03 - 2017-07-09

t

Investigators: Christina Drake

Aim of the experiment: Plasmidisolation of Barnase and colonies 1-5 T7RNAP

Procedure:

  • To get and purified the plasmid
    • protocol from Macherley-Nagel: Plasmid DNA purification, protocol 5: NucleoSpin
    • concentration measurement by nanoprop
  • >03.07.2017
  • tRNA
  • PCR of barnase
  • CDR
  • Check the correct barnasegen
    • 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

    Aim of the 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

    Aim of the experiment: Sequenzingorder barnase

    Procedure:

    • result: sequence was not barnase
  • >16.07.2017
  • tRNA
  • Overnightculture of TyrRS-Heidelberg-plasmid
  • CDR
  • Cellgrowth with the TyrRS-Heidelberg-plasmid
    • 3 ml LB-medium and 0,75 µl chloramphenicol
    • inkubation overnight 37°C, 140rpm

    Transformation of T7-Promotor

    Investigators: Maximilian Edich

    Aim of the experiment: Biobrick Construction with RuBisCo Parts

    Procedure:

    Preculture of colonies with T7-Promotor

    Investigators: Maximilian Edich

    Aim of the experiment: Biobrick Construction with RuBisCo Parts

    Procedure:

    2017-07-17 - 2017-07-23

    Isolate the tRNA

    Investigators: Christina Drake

    Aim of the experiment: PCR of TyrRS-Heidelberg-plasmid

    Procedure:

    Isolation of T7-Promotor

    Investigators: Laura Schlueter

    Aim of the 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

    Aim of the experiment: Biobrick Construction with RuBisCo Parts

    Procedure:

    Biobrickassembly of 2B2 to 2B8, B1 and A1

    Investigators: Maximilian Edich

    Aim of the 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

    Aim of the experiment: Interlab study

    Procedure:

    • dilute test devives (distribution plate 6 & 7) in 10 ֌ ddH20
    • Transformation

    Transformation of 2B2-2B8, B1 and A1

    Investigators: Maximilian Edich

    Aim of the experiment: Biobrick Construction with RuBisCo Parts

    Procedure:

    Plasmidisolation of 2B1

    Investigators: Maximilian Edich

    Aim of the experiment: Biobrick Construction with RuBisCo Parts

    Procedure:

    • Nucleospin Plasmidisolation protocol
      • got 47 ng/µl and 41 ng/µl

    Reverse the insert

    Investigators: Christina Drake

    Aim of the 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

    Aim of the experiment: PCR of T7GFP

    Procedure:

    Preparation of over-night culture

    Investigators: Camilla Maerz

    Aim of the 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 (37у, 200 rpm)

    2017-07-24 - 2017-07-30

    Repeat the degestion and transformation from the 18th and 19th july

    Investigators: Maximilian Edich

    Aim of the experiment: Biobrick Construction with RuBisCo Parts

    Procedure:

    Cellgrowth with the plasmid

    Investigators: Christina Drake

    Aim of the experiment: Overnightculture of tRNA-psB1C3

    Procedure:

    • 3 ml LB-medium and 0,75 µl chloramphenicol
    • inkubation overnight 37°C, 140rpm
  • >31.07.2017
  • tRNA
  • Aquacloning of tRNA and backbone
  • CDR
  • construct a biobrick
    • transformation via heatschock

    Cloning of the ori (pSB6A1) in pSB1C3

    Investigators: Laura Schlueter

    Aim of the experiment: aaRS Plasmid (without aaRS)

    Procedure:

    • Gibson Assembly Master Mix
      • Backbone: 3,28 ֌ Ori 1.0: 1,73 ֌
      • Backbone: 0,32 ֌ Ori 1.1: 4,68 ֌
      • Backbone: 0,90 ֌ Ori 3.0: 4,10 ֌
    • Trafo: Hitzeschock
    • not successfull because of the wrong overlapps (try again)

    Colony PCR of the Retrafo of pRS (because of mixed culture)

    Investigators: Laura Schlueter

    Aim of the 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

    Aim of the experiment: aaRS plasmid

    Procedure:

    • PCR with Q5 High-Fidelity Polymerase Master Mix, Primer jb,jn, 65у
    • gelelectrophoresis, 1% Agarose
    • band at 2000-2500 bp (supposed to be around 2200 bp)
    • Clean up from the gel
      • pSB1C3(nur ori): 22,2 ng/֌

    Amplification of pSB1C3 fragments for a 4 part Gibson (see page 4 in the paper-labbook)

    Investigators: Laura Schlueter

    Aim of the experiment: aaRS plasmid

    Procedure:

    • PCR, Q5 Master Mix
      • Primer jj, jn for fragment 2, 65у
      • Primer jb, ht for fragment 4, 65у
    • 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/֌ (overlapp(aaRS)_pSB1C3_overlapp(ori))
      • fragment 4: 19,1 ng/֌ (overlapp(ori)_pSB1C3_overlapp(aaRS))

    Colony PCR of 2B2-2B8, B1 and A1

    Investigators: Maximilian Edich

    Aim of the 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

    Aim of the 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 : stron bands at around 1200 bp
    • because of the the two positive colony PCRs (vf-vr, hq-jk) for these probes, the 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

    Aim of the experiment: aaRS plasmid

    Procedure:

    • Macherey-Nagel purification kit
      • pRS31: 324,1 ng/֌
      • pRS32: 231,8 ng/֌
      • pRS39: 225,5 ng/֌
    • send to the sequencing
    • 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

    plasmid isolation of the positive colonies of the retrafo pRS4,4 (wrong!) and pRS3,12

    Investigators: Laura Schlueter

    Aim of the experiment: aaRS plasmid

    Procedure:

    • Macherey-Nagel purification kit with each 3 mL culture
      • pRS4,4: 44 ng/֌
      • pRS3,12: 68,3 ng/֌
      • not send to sequencing jet

    4 part Gibson and Trafo

    Investigators: Laura Schlueter

    Aim of the experiment: aaRS plasmid

    Procedure:

    • Gibson Assembly Master Mix with 5 ֬ template
      • pSB1C3 fragment (2): 18,5 ng/֌ around 350 bp 2,2 ֌
      • pSB1C3 fragment (4): 19,1 ng/֌ around 1300 bp 0,8 ֌
      • oK1.1 (pMB1 from pSB6A1): 5,8 ng/֌ around 1300 bp 1,8 ֌
      • Tyr-aaRS: 68,5 ng/֌ around 1000 bp 0,2 ֌
    • Trafo via heatshock

    Gibson of the ori in pSB1C3 (pori) and Trafo

    Investigators: Laura Schlueter

    Aim of the experiment: aaRS plasmid

    Procedure:

    • Gibson Assembly Master Mix with 5 ֌ template
      • pSB1C3(nur ori): 22,2 ng/֌ around 2100 bp 1,57 ֌
      • ok1.0: 5,80 ng/֌ around 1258 bp 3,43 ֌
    • Trafo via heatshock

    Transfer positive colonies

    Investigators: Maximilian Edich

    Aim of the 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

    Aim of the experiment: Aquacloning of tRNA and backbone

    Procedure:

    • transformation via heatschock

    construct a biobrick

    Investigators: Christina Drake

    Aim of the experiment: Gibbsonassembly of barnase and backbone

    Procedure:

    • transformation via heatschock
  • >03.08.2017
  • tRNA
  • Overnightculture of tRNA-psB1C3 and barnase-psB1C3
  • CDR
  • Cellgrowth with the plasmid
    • 3 ml LB-medium and 0,75 µl chloramphenicol
    • inkubation overnight 37°C, 140rpm

    Preculture of 2B6, 2B7 and A1

    Investigators: Maximilian Edich

    Aim of the experiment: Biobrick Construction with RuBisCo Parts

    Procedure:

    Plasmidisolation

    Investigators: Maximilian Edich

    Aim of the 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

    Aim of the 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

    Aim of the 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

    Aim of the experiment: aaRS plasmid

    Procedure:

    • PCR Q5 Polymerase (no Master Mix)
      • for 50 ֌ reaction
        • 10 ֌ Reaction Buffer
        • 5 ֌ 2mM dNTPs
        • 1 ֌ Template
        • 0,5 ֌ High Fidelity DNA Polymerase
        • 10,0 ֌ High GC Enhancer
        • 18,5 ֌ ddest H2O
        • 5,0 ֌ Primer
          • pSB1C3 (from RuBisCo, ncAA team): ht, jk 65у
          • aaRS (from Heidelberg) : hq, jk 65у (mistake! should be 58у, redone identically with 58у)
        • digestion of the PCR products with Dpn1: 5 ֌ CutSmart Reaktionsbuffer and 1 ֌ Dpn1 per 50 ֌ PCR product, on 37у 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

    Aim of the 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

    Aim of the experiment: preparation of KRX-competent cells

    Procedure:

    • heatschocktransformation of T7GFP and T7GFP revers in KRX
  • >08.08.2017
  • tRNA
  • PCR of T7RNA-Polymerase
  • CDR
  • construct a biobrick for the selectionplasmid

    • Amplification of the backbone (pRS31) and insert (ori from pSB6A1, oK1.0) for Gibson assembly

    Investigators: Laura Schlueter

    Aim of the experiment: aaRS plasmid

    Procedure:

    • PCR Q5 Polymerase (protocoll of 07.08.2017)
      • pRS31: primer jb, jn 65у
      • oK1: primer jl, jc 65у
    • 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

    Aim of the experiment: aaRS plasmid

    Procedure:

    • agarose-gelelectrophhoresis, 1%
    • purification from the gel with the Macherey-Nagel purification kit
      • oK1.1: 22,5 ng/֌
      • pRS32: 9,5 ng/֌
    • Gibson Assembly Master Mix with:
      • 4,1 ֌ of oK1.1: around 2480 bp 9,5 ng/֌
      • 0,9 ֌ of pRS32: around 1250 bp 22,5 ng/֌
    • Trafo via heatshock

    getting positive clones for further work

    Investigators: Yannic Kerkhoff

    Aim of the 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

    Aim of the experiment: Q5-PCR, gel and PCR-cleanUp of F13

    Procedure:

    • standard Q5-PCR protocol
      • template: K1416000
      • primer fwd: 17hl
      • primer rev: 17mv
      • annealing temperature: 63у
      • extension time: 90 seconds
    • standard PCR-cleanUp protocol
      • 80,6 ng/֌

    Assemble the fragments to get the composite Part BBa_P7:NPA-RS

    Investigators: Yannic Kerkhoff

    Aim of the experiment: Gibson assembly of F13 and NPA-RS gene synthesis

    Procedure:

    getting positive clones for further work

    Investigators: Yannic Kerkhoff

    Aim of the 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

    Aim of the 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: 58у
      • extension time: 50 seconds
    • standard PCR-cleanUp protocol
      • 15,9 ng/֌

    multiplication, verification and purification of the needed fragment F3: StrepLinker1

    Investigators: Yannic Kerkhoff

    Aim of the 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: 61у
      • extension time: 30 seconds
    • standard PCR-cleanUp protocol
      • 17,9 ng/֌

    Biobrickassembly

    Investigators: Maximilian Edich

    Aim of the 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

    Aim of the experiment: Biobrick Construction with RuBisCo Parts

    Procedure:

    Primer annealing preparation

    Investigators: Camilla Maerz

    Aim of the experiment: M.A.X restriction system - Primer annealing test

    Procedure:

    • Both Primers (17jh; 17ji) were resuspended in 1x Composite Buffer
    • Dilutions: 100 ֍, 10 ֍, 5 ֍, 2.5 ֍, 0.5 ֍
    • OD 260 were measured to determine the real concentration

    2017-08-14 - 2017-08-20

    Screening for positive transformations containing the Biobrick BBa_P2:GLS2

    Investigators: Yannic Kerkhoff

    Aim of the experiment: GoTaq PCR of 4 clones

    Procedure:

    • standard GoTaq-protocol
      • template: colonie pcr clones of GA_P1:GLS1
      • primer fwd: Pr姩x_fwd
      • primer rev: Suffix_rev
      • annealing temperature: 56у
      • extension time: 40 seconds
    • positive clone 3

    Primer annealing of 17jh & 17ji

    Investigators: Camilla Maerz

    Aim of the experiment: M.A.X restriction system - Primer annealing test

    Procedure:

    • Protocol for annealing oligonucleotides (sigmaaldrich)
      • Eqimolar Primers were mixed (50 ֌)
      • Thermal profile (thermocycler)
        • 95 у, 2 min
        • Cool to 25 у over 45 min
        • Cool to 4 у for temporary storage

    Primer annealing of 17jl & 17jm

    Investigators: Camilla Maerz

    Aim of the experiment: M.A.X restriction system - Primer annealing test

    Procedure:

    • Both Primers were resuspended in ddH20 to a final concentration of 100 ֍
    • HEPES annealing standard protocol
    • Aqua annealing standard protocol
      • Final volume: 20 ֌
    • Agarose gel electrophoresis (2%)
      • 60 V, 50 min

    Native PAGE of annealed primers (17jl & 17jm)

    Investigators: Camilla Maerz

    Aim of the experiment: M.A.X restriction system - Primer annealing test

    Procedure:

    • Native DNA PAGE standard protocol
      • tested samples:
        • HEPES annealing (17jl & 17jm): 100 ֍, 10 ֍, 5 ֍, 2.5 ֍, 1 ֍, 0.5 ֍
        • Aqua annealing (17jl & 17jm): 100 ֍, 10 ֍, 5 ֍, 2.5 ֍, 1 ֍, 0.5 ֍
      • Variation: ~40 mA

    2017-08-21 - 2017-08-27

    Native PAGE of annealed primers (17jl & 17jm)

    Investigators: Camilla Maerz

    Aim of the experiment: M.A.X restriction system - Primer annealing test

    Procedure:

    • Native DNA PAGE standard protocol
      • tested samples:
        • HEPES annealing (17jl & 17jm): 100 ֍, 10 ֍, 5 ֍, 2.5 ֍, 1 ֍, 0.5 ֍
        • Aqua annealing (17jl & 17jm): 100 ֍, 10 ֍, 5 ֍, 2.5 ֍, 1 ֍, 0.5 ֍
      • Variation: 100 V, 1 h

    Native PAGE of annealed primers (17jl & 17jm)

    Investigators: Camilla Maerz

    Aim of the experiment: M.A.X restriction system - Primer annealing test

    Procedure:

    • Native DNA PAGE standard protocol
      • tested samples:
        • HEPES annealing (17jl & 17jm): 100 ֍, 10 ֍, 5 ֍, 2.5 ֍, 1 ֍, 0.5 ֍
        • Aqua annealing (17jl & 17jm): 100 ֍, 10 ֍, 5 ֍, 2.5 ֍, 1 ֍, 0.5 ֍
      • Variation: 60 V, 2 h

    Native Page of annealed primers (17jl & 17jm; 17jh & 17ji)

    Investigators: Camilla Maerz

    Aim of the experiment: M.A.X restriction system - Primer annealing test

    Procedure:

    • Native DNA PAGE standard protocol
      • tested samples:
        • HEPES annealing (17jl & 17jm): 0.5 ֍
        • Aqua annealing (17jl & 17jm): 0.5 ֍
        • Composite Buffer annealing (17jh & 17 jm): 0.5 ֍
        • all Primers (ssDNA): 1 ֍

    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

    Aim of the experiment: M.A.X restriction system - Primer annealing test

    Procedure:

    • all samples were diluted to a final concentration of 0.5 ֍
    • Native DNA PAGE standard protocol

    Restriction digest of control annealings (mutC)

    Investigators: Camilla Maerz

    Aim of the experiment: M.A.X restriction system

    Procedure:

    • Restriction Digest (MnlI) of aqua annealing sample: mutC (0.5 ֍)
      • 2 ֌ MnlI
      • 5 ֌ CutSmart
      • 25 ֌ mutC dsDNA
      • 18 ֌ nuclease-free H2O
      • Incubation: 37 у, 1 h
      • Inactivation: 20 min., 65 у
    • Native DNA PAGE standard protocol
      • Tested samples:
        • digested mutC sample
        • native mutC sample
        • ssDNA primers (17jl & 17jm)

    Restriction digest of aqua & HEPES annealing

    Investigators: Camilla Maerz

    Aim of the experiment: M.A.X restriction system

    Procedure:

    • Restriction Digest (MnlI) of aqua & HEPES annealing sample: mutC (0.5 ֍)
      • 2 ֌ MnlI
      • 5 ֌ CutSmart
      • 25 ֌ mutC dsDNA
      • 18 ֌ nuclease-free H2O
      • Incubation: 37 у, 1 h
      • Inactivation: 20 min., 65 у
    • 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

    Aim of the experiment: M.A.X restriction system

    Procedure:

    • Aqua annealing standard protocol (final concentration: 0.5 ֍)
      • mutA: 17jf & 17jg
      • mutT: 17jj & 17jk
      • mutG: 17jh & 17ji
      • mutC: 17jl & 17jm
    • final volume of each sample: 50 ֌

    2017-09-04 - 2017-09-10

    Restriction digest of control annealings (mutA, mutT, mutG, mutC) and aqua annealing of oligo2

    Investigators: Camilla Maerz

    Aim of the experiment: M.A.X restriction system

    Procedure:

    • Oligo2 annealing
      • Aqua annealing standard protocol (final concentration: 0.5 ֍)
    • Restricted samples:
      • MnlI -> mutC, oligo2
      • SapI -> mutG, oligo2
      • BsaI -> mutT, oligo2
      • EciI -> mutA, oligo2
    • Restriction Digest:
      • 1 ֌ Enzyme
      • 5 ֌ CutSmart
      • 25 ֌ mutC dsDNA
      • 19 ֌ nuclease-free H2O
      • Incubation: 37 у, 1 h
      • Inactivation: 20 min., 65 у
    • Native DNA PAGE standard protocol

    2017-09-11 - 2017-09-17

    Annealing, restriction digest and native PAGE of all samples

    Investigators: Camilla Maerz

    Aim of the experiment: M.A.X restriction system

    Procedure:

    • Aqua annealing standard protocol (final concentration: 0.5 ֍)
      • mutA: 17jf & 17jg
      • mutT: 17jj & 17jk
      • mutG: 17jh & 17ji
      • mutC: 17jl & 17jm
      • oligo2: olgio2_sense & oligo2_antisense
    • final volume of each sample: 50 ֌
    • Restricted samples:
      • MnlI -> mutC, oligo2
      • SapI -> mutG, oligo2
      • BsaI -> mutT, oligo2
      • EciI -> mutA, oligo2
    • Restriction Digest:
      • 1 ֌ Enzyme
      • 5 ֌ CutSmart
      • 25 ֌ mutC dsDNA
      • 19 ֌ nuclease-free H2O
      • Incubation: 37 у, 1 h
      • Inactivation: 20 min., 65 у
    • Native DNA PAGE standard protocol

    Annealing, restriction digest and native PAGE of all samples

    Investigators: Camilla Maerz

    Aim of the experiment: M.A.X restriction system

    Procedure:

    • Aqua annealing standard protocol (final concentration: 0.5 ֍)
      • mutA: 17jf & 17jg
      • mutT: 17jj & 17jk
      • mutG: 17jh & 17ji
      • mutC: 17jl & 17jm
      • oligo2: olgio2_sense & oligo2_antisense
    • final volume of each sample: 2x50 ֌
    • Restricted samples:
      • MnlI -> mutC
      • SapI -> mutG, oligo2
      • BsaI -> mutT
      • EciI -> mutA
    • Restriction Digest:
      • 1 ֌ Enzyme
      • 5 ֌ CutSmart
      • 25 ֌ mutC dsDNA
      • 19 ֌ nuclease-free H2O
      • Incubation: 37 у, 1 h
      • Inactivation: 20 min., 65 у
    • Native DNA PAGE standard protocol

    2017-09-18 - 2017-09-24

    -Agarose gel electrophoresis (2%)

    Investigators: Agarose, gel, electrophoresis, of, digested, mutA,, mutT,, mutG, &, mutC

    Aim of the experiment: CMZ

    Procedure:

    • 60 V, 40 min

    Annealing, restriction digest and native PAGE of all samples

    Investigators: Camilla Maerz

    Aim of the experiment: M.A.X restriction system

    Procedure:

    • Aqua annealing standard protocol (final concentration: 0.5 ֍)
      • mutA: 17jf & 17jg
      • mutT: 17jj & 17jk
      • mutG: 17jh & 17ji
      • mutC: 17jl & 17jm
      • oligo2: olgio2_sense & oligo2_antisense
    • final volume of each sample: 2x50 ֌
    • Restricted samples:
      • MnlI -> mutC
      • SapI -> mutG, oligo2
      • BsaI -> mutT
      • EciI -> mutA
    • Restriction Digest:
      • 1 ֌ Enzyme
      • 5 ֌ CutSmart
      • 25 ֌ mutC dsDNA
      • 19 ֌ nuclease-free H2O
      • Incubation: 37 у, 1 h
      • Inactivation: 20 min., 65 у
    • Native DNA PAGE standard protocol

    Colony PCR of pSB3K5_mRFP_link_ccdB and pSB1C3_PtNTT2(31-575)-GFP

    Investigators: Camilla Maerz

    Aim of the experiment: Construction of pSB1C3-PlacUV5_PtNTT2

    Procedure:

    • PCR
      • Go Tag (Promega) protocol
      • Primer: VR, VF2
      • Danaturation: 56 у
      • Elongation: 90 s & 180 s
    • Agarose gel electrophoresis (1%)
      • 100 V, 30 min

    2017-09-25 - 2017-10-01

    -Aqua annealing standard protocol (final concentration: 0.5 ֍)

    Investigators: Aqua, annealing, of, mutA-1,, mutG-1,, mutC-1,, mutG-1, &, oligo1

    Aim of the experiment: ??

    Procedure:

    • mutA-1: 17iv & 17iw
      • mutT-1: 17jb & 17jc
      • mutG-1: 17ix & 17iy
      • mutC-1: 17jd & 17jc
      • oligo1: olgio1_sense & oligo1_antisense
    • final volume: 50 ֌

    2017-10-02 - 2017-10-08

    UBP-PCR with TiTaq

    Investigators: Camilla Maerz

    Aim of the experiment: PCR with UBP

    Procedure:

    • PCR
      • PCR-UBP standard protocol (TiTaq)
      • samples:
        • Ligation of: mutA, mutT, mutG, mutC and oligo 2 (1 ng/֌)
      • Primer: 17vt & 17vu
      • Primer annealing: 56 у
      • Elongation: 20 s
    • PCR and DNA purification kit (Macherey-Nagel)
    • Restriction Digest
      • 1 ֌ Enzyme
      • 5 ֌ CutSmart
      • 25 ֌ mutC dsDNA
      • 19 ֌ nuclease-free H2O
      • Incubation: 37 у, 1 h
      • Inactivation: 20 min., 65 у
    • Restricted samples:
      • MnlI -> mutC
      • SapI -> mutG
      • BsaI -> mutT
      • EciI -> mutA
      • EciI, BsaI, SapI, MnlI -> oligo2
      • Variation: oligo2 was digested with 0.5 ֌ of each enzyme
    • Agarose gel electrophoresis (2 %)
      • 100 V, 20 min

    UBP-PCR with GoTaq G2 and TiTaq

    Investigators: Camilla Maerz

    Aim of the 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/֌)
      • Primer: 17 vt & 17 vu
      • Primer annealing: 56 у
      • Elongation: 20 s
    • PCR with GoTaq:
      • PCR-UBP standard protocol (TiTaq)
      • samples:
        • Ligation of: mutA, mutT, mutG and mutC (5 ng/֌)
      • Primer: VR & VF2
      • Primer annealing: 56 у
      • Elongation: 20 s
    • PCR with GoTaq:
      • PCR-UBP standard protocol (TiTaq)
      • samples:
        • Ligation of: mutA, mutT, mutG, mutC and oligo2 (5 ng/֌)
      • Primer: 17 vt & 17 vu
      • Primer annealing: 56 у
      • Elongation: 20 s

    Restriction digest of ligated oligo2

    Investigators: Camilla Maerz

    Aim of the experiment: PCR with UBP

    Procedure:

    • Restriction Digest
      • 5 ֌ CutSmart
      • 30 ֌ ligated oligo2 DNA
      • 2 ֌ EcoRV
      • filled up to final volume 50 ֌ with ddH20
      • Incubation: 120 min, 37 у
      • Inactivation: 20 min, 65 у

    2917-06-14 - 2917-06-20

    Duplicate the KRX-e.coli

    Investigators: Christina Drake, Denise Kerkhoff

    Aim of the experiment: Plated out: KRX-e.coli as glycostocsolution

    Procedure:

    • plated out on kanamycin
    • incubation at 37°C