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<h1>Tuesday, June 10th.</h1> | <h1>Tuesday, June 10th.</h1> | ||
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<h1>Wednesday, June 11th.</h1> | <h1>Wednesday, June 11th.</h1> | ||
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<h1>Thursday, June 12th.</h1> | <h1>Thursday, June 12th.</h1> | ||
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<h1>Friday, June 13th.</h1> | <h1>Friday, June 13th.</h1> | ||
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<h1>Monday, June 16th.</h1> | <h1>Monday, June 16th.</h1> | ||
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<h1>Wednesday, June 18th.</h1> | <h1>Wednesday, June 18th.</h1> | ||
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<h1>Friday, June 20th.</h1> | <h1>Friday, June 20th.</h1> | ||
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<h1>Monday, June 23rd.</h1> | <h1>Monday, June 23rd.</h1> | ||
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<div id="date-6-24"> | <div id="date-6-24"> | ||
<h1>Tuesday, June 24th.</h1> | <h1>Tuesday, June 24th.</h1> | ||
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<h1>Thursday, June 26th.</h1> | <h1>Thursday, June 26th.</h1> | ||
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<div id="date-6-27"> | <div id="date-6-27"> | ||
<h1>Friday, June 27th.</h1> | <h1>Friday, June 27th.</h1> | ||
− | + | <li> Cara Jones</li> | |
− | + | <ul>Plasmid Resuspension, Primer Dilutions, PCR: <a href="https://static.igem.org/mediawiki/2017/b/b7/T--Georgia_State--6_27_17_cj.pdf"> See attached procedure for buffer recipes and instructions.</a> | |
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− | </div> | + | |
<div id="date-6-28"> | <div id="date-6-28"> | ||
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<h1>Friday, July 4th.</h1> | <h1>Friday, July 4th.</h1> | ||
<ul> | <ul> | ||
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<div id="date-7-6"> | <div id="date-7-6"> | ||
<h1>Thursday, July 6th.</h1> | <h1>Thursday, July 6th.</h1> | ||
+ | <li> Cara Jones</li> | ||
+ | <ul>Ligation of Mamba insert+pGEX vector and DNA clean up of pGEX+mamba restriction digest: <a href="https://static.igem.org/mediawiki/2017/0/0d/T--Georgia_State--7_6_17_cj.pdf"> See attached procedure for instructions.</a> | ||
</div> | </div> | ||
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<ul><b>Procedure:</b><a href="https://static.igem.org/mediawiki/2017/0/0a/T--Georgia_State--APTestprotocol.pdf">See attached procedure.</a></ul> | <ul><b>Procedure:</b><a href="https://static.igem.org/mediawiki/2017/0/0a/T--Georgia_State--APTestprotocol.pdf">See attached procedure.</a></ul> | ||
<ul><b>Results:</b> The Ap spread outward but stayed confined meaning the crosslinking worked. Adding the BCIP solution to the tube with the paper produced a noticeable discoloration. Adding the enzyme to the BCIP only produced a small discoloration. The binding of the alkaline phosphatase and BCIP to the paper was successful. The reaction worked only in the intended areas meaning that if the AP is bound to the factor c it may produce a signal only if it is cut, the AP nor BCIP disperses in the solution after being bound to the paper.<br><img src="https://static.igem.org/mediawiki/2017/c/c5/T--Georgia_State--APTest.png"></ul> | <ul><b>Results:</b> The Ap spread outward but stayed confined meaning the crosslinking worked. Adding the BCIP solution to the tube with the paper produced a noticeable discoloration. Adding the enzyme to the BCIP only produced a small discoloration. The binding of the alkaline phosphatase and BCIP to the paper was successful. The reaction worked only in the intended areas meaning that if the AP is bound to the factor c it may produce a signal only if it is cut, the AP nor BCIP disperses in the solution after being bound to the paper.<br><img src="https://static.igem.org/mediawiki/2017/c/c5/T--Georgia_State--APTest.png"></ul> | ||
+ | <li> Cara Jones</li> | ||
+ | <ul>Colony PCR: <a href="https://static.igem.org/mediawiki/2017/2/20/T--Georgia_State--7_7_17_cj.pdf"> See attached procedure for instructions.</a> | ||
</div> | </div> | ||
<div id="date-7-8"> | <div id="date-7-8"> | ||
− | <h1> | + | <h1>Saturday, July 8th.</h1> |
</div> | </div> | ||
<div id="date-7-9"> | <div id="date-7-9"> | ||
− | <h1> | + | <h1>Sunday, July 9th.</h1> |
</div> | </div> | ||
<div id="date-7-10"> | <div id="date-7-10"> | ||
− | <h1> | + | <h1>Monday, July 10th.</h1> |
− | + | <li> Cara Jones</li> | |
− | + | <ul>Restriction digest of mamba in pGEX: <a href="https://static.igem.org/mediawiki/2017/e/ed/T--Georgia_State--7_10_17_cj.pdf"> See attached procedure for instructions.</a> | |
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</div> | </div> | ||
<div id="date-7-11"> | <div id="date-7-11"> | ||
− | <h1> | + | <h1>Tuesday, July 11th.</h1> |
+ | <li> Cara Jones</li> | ||
+ | <ul>Sequencing results from macrogen and planning : <a href="https://static.igem.org/mediawiki/2017/c/c7/T--Georgia_State--7_11_17_cj.pdf"> See attached.</a> | ||
</div> | </div> | ||
<div id="date-7-12"> | <div id="date-7-12"> | ||
− | <h1> | + | <h1>Wednesday, July 12th.</h1> |
</div> | </div> | ||
<div id="date-7-13"> | <div id="date-7-13"> | ||
− | <h1> | + | <h1>Thursday, July 13th.</h1> |
+ | <li> Cara Jones</li> | ||
+ | <ul>Transformation of mamba in pGEX into Bl21 Competent cells: <a href="https://static.igem.org/mediawiki/2017/5/5a/T--Georgia_State--7_13_17_cj.pdf"> See attached procedure for instructions.</a> | ||
</div> | </div> | ||
<div id="date-7-14"> | <div id="date-7-14"> | ||
− | <h1> | + | <h1>Friday, July 14th.</h1> |
</div> | </div> | ||
<div id="date-7-15"> | <div id="date-7-15"> | ||
− | <h1> | + | <h1>Saturday, July 15th.</h1> |
− | + | </div> | |
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− | </div> | + | |
<div id="date-7-16"> | <div id="date-7-16"> | ||
− | <h1> | + | <h1>Sunday, July 16th.</h1> |
− | + | </div> | |
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<div id="date-7-17"> | <div id="date-7-17"> | ||
− | <h1> | + | <h1>Monday, July 17th.</h1> |
+ | <li> Cara Jones</li> | ||
+ | <ul>Interlab 2017 transformations and Mamba in pGEX protein expression: <a href="https://static.igem.org/mediawiki/2017/c/c7/T--Georgia_State--7_17_17_cj.pdf"> See attached procedure for instructions.</a> | ||
</div> | </div> | ||
<div id="date-7-18"> | <div id="date-7-18"> | ||
− | <h1> | + | <h1>Tuesday, July 18th.</h1> |
</div> | </div> | ||
<div id="date-7-19"> | <div id="date-7-19"> | ||
− | <h1> | + | <h1>Wednesday, July 19th.</h1> |
− | + | </div> | |
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<div id="date-7-20"> | <div id="date-7-20"> | ||
− | <h1> | + | <h1>Thursday, July 20th.</h1> |
</div> | </div> | ||
<div id="date-7-21"> | <div id="date-7-21"> | ||
− | <h1> | + | <h1>Friday, July 21st.</h1> |
</div> | </div> | ||
<div id="date-7-22"> | <div id="date-7-22"> | ||
− | <h1> | + | <h1>Saturday, July 22nd.</h1> |
− | + | </div> | |
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<div id="date-7-23"> | <div id="date-7-23"> | ||
− | <h1> | + | <h1>Sunday, July 23rd.</h1> |
</div> | </div> | ||
<div id="date-7-24"> | <div id="date-7-24"> | ||
− | <h1> | + | <h1>Monday, July 24th.</h1> |
</div> | </div> | ||
<div id="date-7-25"> | <div id="date-7-25"> | ||
− | <h1> | + | <h1>Tuesday, July 25th.</h1> |
</div> | </div> | ||
<div id="date-7-26"> | <div id="date-7-26"> | ||
− | <h1> | + | <h1>Wednesday, July 26th.</h1> |
− | + | </div> | |
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<div id="date-7-27"> | <div id="date-7-27"> | ||
− | <h1> | + | <h1>Thursday, July 27th.</h1> |
+ | <li> Cara Jones</li> | ||
+ | <ul>Dilution of Mamba biobrick prefix/suffix, PCR of mamba in pGEX, PCR purification: <a href="https://static.igem.org/mediawiki/2017/7/7e/T--Georgia_State--7_27_17_cj.pdf"> See attached procedure for instructions.</a> | ||
</div> | </div> | ||
<div id="date-7-28"> | <div id="date-7-28"> | ||
− | <h1> | + | <h1>Friday, July 28th.</h1> |
</div> | </div> | ||
<div id="date-7-29"> | <div id="date-7-29"> | ||
− | <h1> | + | <h1>Saturday, July 29th.</h1> |
</div> | </div> | ||
<div id="date-7-30"> | <div id="date-7-30"> | ||
− | <h1> | + | <h1>Sunday, July 30th.</h1> |
</div> | </div> | ||
<div id="date-7-31"> | <div id="date-7-31"> | ||
− | <h1> | + | <h1>Monday, July 31st.</h1> |
</div> | </div> | ||
− | <!-- | + | <!-- August --> |
<div id="date-8-1"> | <div id="date-8-1"> | ||
<h1>Tuesday,August 1st.</h1> | <h1>Tuesday,August 1st.</h1> | ||
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<ul><b>Results:</b><table class="w3-table"><tr><td><img src="https://static.igem.org/mediawiki/2017/6/62/T--Georgia_State--8.3DoubleRestrictionDigestHCG.jpg"></td><td>Since the diagnostic gel was a success the PSB1C backbone was extracted from the Lt 10 plasmid using an E-gel.</td></tr></table> </ul> | <ul><b>Results:</b><table class="w3-table"><tr><td><img src="https://static.igem.org/mediawiki/2017/6/62/T--Georgia_State--8.3DoubleRestrictionDigestHCG.jpg"></td><td>Since the diagnostic gel was a success the PSB1C backbone was extracted from the Lt 10 plasmid using an E-gel.</td></tr></table> </ul> | ||
</div> | </div> | ||
+ | |||
<div id="date-8-6"> | <div id="date-8-6"> | ||
<h1>Sunday, August 6th.</h1> | <h1>Sunday, August 6th.</h1> | ||
</div> | </div> | ||
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<div id="date-8-7"> | <div id="date-8-7"> | ||
<h1>Monday, August 7th.</h1> | <h1>Monday, August 7th.</h1> | ||
+ | <li> Cara Jones</li> | ||
+ | <ul><b>Objective:</b>Culture of transformed E.coli with mamba in PGEX</ul> | ||
+ | <ul>I picked 10 colonies from plates and did overnight cultures in LB</ul> | ||
</div> | </div> | ||
<div id="date-8-8"> | <div id="date-8-8"> | ||
<h1>Tuesday, August 8th.</h1> | <h1>Tuesday, August 8th.</h1> | ||
+ | <li> Eudoxie Bataba</li> | ||
+ | <ul><b>Objective:</b>I will get the mamba in PGEX plasmid from transformed E.coli</ul> | ||
+ | <ul><b>Materials:</b>NEB miniprep kit, 50% glycerol</ul> | ||
+ | <ul><b>Equipment:</b>Centrifuge and Nanophotometer</ul> | ||
+ | <ul><b>Procedure:</b><a href="https://static.igem.org/mediawiki/2017/f/ff/T--Georgia_State--mamba_miniprep.jpg">See attached procedure.</a></ul> | ||
+ | <ul><b>Results:</b> <table class="w3-table"> | ||
+ | <tr> | ||
+ | <th>Sample</th> | ||
+ | <th>Concentration (ng/uL)</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>1</td> | ||
+ | <td>118</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>2</td> | ||
+ | <td> 123</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>3</td> | ||
+ | <td> 85</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>4</td> | ||
+ | <td>133</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>5</td> | ||
+ | <td>90</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>6</td> | ||
+ | <td> 75</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>7</td> | ||
+ | <td> 72</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>8</td> | ||
+ | <td> 100</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>9</td> | ||
+ | <td> 65</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>10</td> | ||
+ | <td> 140</td> | ||
+ | </tr> | ||
+ | </table></ul> | ||
+ | <br> | ||
+ | <li> Cara Jones</li> | ||
+ | <ul>SDS PAGE and Coomassie gel stain of purified mamba in pGEX: <a href="https://static.igem.org/mediawiki/2017/2/28/T--Georgia_State--8_8_17_cj.pdf"> See attached procedure for instructions.</a> | ||
</div> | </div> | ||
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<div id="date-8-10"> | <div id="date-8-10"> | ||
− | <h1>Thursday, August | + | <h1>Thursday, August 10th.</h1> |
<li> Tori Radcliff </li> | <li> Tori Radcliff </li> | ||
<ul> Transformation of hCG-PSB1C3 ligation and overnight culture </uL> | <ul> Transformation of hCG-PSB1C3 ligation and overnight culture </uL> | ||
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<div id="date-8-11"> | <div id="date-8-11"> | ||
− | <h1>Friday, August | + | <h1>Friday, August 11th.</h1> |
<li>Tori Radcliff</li> | <li>Tori Radcliff</li> | ||
<ul>Colony PCR of hCG-PSB1C3 colonies</ul> | <ul>Colony PCR of hCG-PSB1C3 colonies</ul> | ||
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<div id="date-8-12"> | <div id="date-8-12"> | ||
− | <h1>Saturday, August | + | <h1>Saturday, August 12th.</h1> |
</div> | </div> | ||
<div id="date-8-13"> | <div id="date-8-13"> | ||
− | <h1>Sunday, August 13th</h1> | + | <h1>Sunday, August 13th.</h1> |
<li> Tori Radcliff</li> | <li> Tori Radcliff</li> | ||
<ul>Overnight culture of hCG-PSB1C3 colonies</ul> | <ul>Overnight culture of hCG-PSB1C3 colonies</ul> | ||
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<div id="date-8-14"> | <div id="date-8-14"> | ||
− | <h1>Monday, August 14th</h1> | + | <h1>Monday, August 14th.</h1> |
<li>Tori Radcliff</li> | <li>Tori Radcliff</li> | ||
<ul> Miniprep of hCG-PSB1C3 cultures </ul> | <ul> Miniprep of hCG-PSB1C3 cultures </ul> | ||
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<ul><b>Objective:</b> Factor C was divided into 4 G-blocks and ordered from IDT. A PCR was performed to add overlaps to each block so that subsequent overlap extension PCRs could be performed.</ul> | <ul><b>Objective:</b> Factor C was divided into 4 G-blocks and ordered from IDT. A PCR was performed to add overlaps to each block so that subsequent overlap extension PCRs could be performed.</ul> | ||
<ul><b>Procedure:</b> <a href="">See attached protocol.</a></ul> | <ul><b>Procedure:</b> <a href="">See attached protocol.</a></ul> | ||
− | <ul><b>Results:</b><table class="w3-table"><tr><td><img | + | <ul><b>Results:</b><table class="w3-table"><tr><td><img src="https://static.igem.org/mediawiki/2017/0/09/T--Georgia_State--overlappcr1.png"></td></tr><tr><td>A simulated gel was created and then the actual sampled of each were run on an e-gel. 5ul of product with 1ul of 6X dye and 14 ul of DI water was added to each well. |
+ | The wells include the following from left to right: Gene Ruler 1kb DNA ladder, PCR of g-block 1, PCR of g-block 2, PCR of g-block 3, PCR of g-block 4. | ||
+ | </td></tr></table> | ||
</ul> | </ul> | ||
+ | <li> Cara Jones</li> | ||
+ | <ul>Re-do Restrction Digest of mamba: <a href="https://static.igem.org/mediawiki/2017/b/bd/T--Georgia_State--8_21_17_cj.pdf"> See attached procedure for instructions.</a> | ||
</div> | </div> | ||
<div id="date-8-15"> | <div id="date-8-15"> | ||
− | <h1> | + | <h1>Tuesday, August 15th.</h1> |
+ | <li> Tori Radcliff</li> | ||
+ | <ul> Overlap Extension PCR</ul> | ||
+ | <ul><b>Objective:</b>To combine the four PCR-ed g-blocks of factor-C.</ul> | ||
+ | <ul><b>Procedure:</b> <a href="https://static.igem.org/mediawiki/2017/1/13/T--Georgia_State--OverlapPCRprotocol.pdf">See attached protocol for details</a></ul> | ||
+ | <ul><b>Results:</b><table class="w3-table"><tr><td><img src="https://static.igem.org/mediawiki/2017/0/08/T--Georgia_State--8.15SimulatedgelofOVERLAP.jpg"></td><td><img src="https://static.igem.org/mediawiki/2017/c/ca/T--Georgia_State--8.15gelofOVERLAP.jpg"></td></tr><tr><td>Simulated gel</td><td>Wells contain the following: | ||
+ | Gene Ruler 1kb plus MW ladder, Reaction of blocks 1 and 2, Negative control (blocks 1 and 2), Reaction of blocks 3 and 4, Negative control (blocks 3 and 4), Reaction of 1 and 2 with 3 and 4 products, negative control (reactions 1 and 2 with 3 and 3 and 4), and Gene Ruler 1kb plus MW ladder. | ||
+ | </td></tr></table></ul> | ||
</div> | </div> | ||
<div id="date-8-16"> | <div id="date-8-16"> | ||
− | <h1> | + | <h1>Wednesday, August 16th.</h1> |
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</div> | </div> | ||
<div id="date-8-17"> | <div id="date-8-17"> | ||
− | <h1> | + | <h1>Thursday, August 17th.</h1> |
<li>Tori Radcliff</li> | <li>Tori Radcliff</li> | ||
<ul><b>Objective:</b>Prepare hCG for ligation into pGEX through PCR </ul> | <ul><b>Objective:</b>Prepare hCG for ligation into pGEX through PCR </ul> | ||
Line 690: | Line 580: | ||
</ul> | </ul> | ||
<ul><b>Equipment:</b>Thermocycler</ul> | <ul><b>Equipment:</b>Thermocycler</ul> | ||
− | <ul><b>Procedure:</b> | + | <ul><b>Procedure:</b>Two 50 ul PCR reactions were performed. 25ul of qiagen mastermix, 2ul of block 5 DNA, 2.5ul of forward primer, 2.5 ul of reverse primer and 18 ul of nuclease-free water was added to tube 1 and tube 2. </ul> |
− | </ | + | <li> Tatenda Tela</li> |
− | + | <ul> Overnight cultures</ul> | |
− | < | + | <ul> Colony PCR of Cara's transformants</ul> |
− | + | ||
</div> | </div> | ||
<div id="date-8-18"> | <div id="date-8-18"> | ||
− | <h1> | + | <h1>Friday, August 18th.</h1> |
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<div id="date-8-19"> | <div id="date-8-19"> | ||
− | <h1> | + | <h1>Saturday, August 19th.</h1> |
− | + | </div> | |
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− | </div> | + | |
<div id="date-8-20"> | <div id="date-8-20"> | ||
− | <h1> | + | <h1>Sunday, August 20th.</h1> |
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</div> | </div> | ||
<div id="date-8-21"> | <div id="date-8-21"> | ||
− | <h1> | + | <h1>Monday, August 21st.</h1> |
− | + | <li>Tatenda Tela</li> | |
− | + | <ul> Miniprep</ul> | |
− | + | <ul>Ligations</ul> | |
− | + | ||
− | + | </div> | |
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− | </div> | + | |
<div id="date-8-22"> | <div id="date-8-22"> | ||
− | <h1> | + | <h1>Tuesday, August 22nd.</h1> |
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</div> | </div> | ||
<div id="date-8-23"> | <div id="date-8-23"> | ||
− | <h1> | + | <h1>Wednesday, August 23rd.</h1> |
− | + | <li> Cara Jones</li> | |
− | + | <ul>Diagnostic digest and electrophoresis of mamba: <a href="https://static.igem.org/mediawiki/2017/0/0b/T--Georgia_State--8_23_17_cj.pdf"> See attached procedure for instructions.</a> | |
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</div> | </div> | ||
<div id="date-8-24"> | <div id="date-8-24"> | ||
− | <h1> | + | <h1>Thursday, August 24th.</h1> |
− | + | <li> Cara Jones</li> | |
− | + | <ul>Site directed mutagenesis of mambalgin: <a href="https://static.igem.org/mediawiki/2017/6/62/T--Georgia_State--8_24_17_cj.pdf"> See attached procedure for instructions.</a> | |
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</div> | </div> | ||
<div id="date-8-25"> | <div id="date-8-25"> | ||
− | <h1> | + | <h1>Friday, August 25th.</h1> |
<li>Tori Radcliff</li> | <li>Tori Radcliff</li> | ||
<ul><b>Objective:</b>To repeat the double digest of PSCB1C3 and hCG PCR</ul> | <ul><b>Objective:</b>To repeat the double digest of PSCB1C3 and hCG PCR</ul> | ||
Line 854: | Line 634: | ||
<ul><b>Equipment:</b>Thermocycler</ul> | <ul><b>Equipment:</b>Thermocycler</ul> | ||
<ul><b>Procedure:</b><a href="https://static.igem.org/mediawiki/2017/0/02/T--Georgia_State--hCGdigestprotocol.pdf">See attached procedure.</a></ul> | <ul><b>Procedure:</b><a href="https://static.igem.org/mediawiki/2017/0/02/T--Georgia_State--hCGdigestprotocol.pdf">See attached procedure.</a></ul> | ||
+ | <li> Cara Jones</li> | ||
+ | <ul>Diagnostic gel of mutanagized mamba: <a href="https://static.igem.org/mediawiki/2017/5/54/T--Georgia_State--8_25_17_cj.pdf"> See attached procedure for instructions.</a> | ||
</div> | </div> | ||
<div id="date-8-26"> | <div id="date-8-26"> | ||
− | <h1> | + | <h1>Saturday, August 26th.</h1> |
</div> | </div> | ||
<div id="date-8-27"> | <div id="date-8-27"> | ||
− | <h1> | + | <h1>Sunday, August 27th.</h1> |
− | + | </div> | |
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<div id="date-8-28"> | <div id="date-8-28"> | ||
− | <h1> | + | <h1>Monday, August 28th.</h1> |
− | + | <li> Cara Jones</li> | |
− | + | <ul>Transformation of Mutagenesis RxNs: <a href="https://static.igem.org/mediawiki/2017/4/4e/T--Georgia_State--8_28_17_cj.pdf"> See attached procedure for instructions.</a> | |
− | + | </div> | |
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− | </div> | + | |
<div id="date-8-29"> | <div id="date-8-29"> | ||
− | <h1> | + | <h1>Tuesday, August 29th.</h1> |
<li>Tori Radcliff</li> | <li>Tori Radcliff</li> | ||
+ | <ul> Ligation of hCG and PSB1C3 | ||
<ul><b>Objective:</b>To ligate hCG and PSB1C3</ul> | <ul><b>Objective:</b>To ligate hCG and PSB1C3</ul> | ||
<ul><b>Materials:</b> digested vector cleaned by Cara Jones, T4 ligase and buffer, digested hCG | <ul><b>Materials:</b> digested vector cleaned by Cara Jones, T4 ligase and buffer, digested hCG | ||
</ul> | </ul> | ||
− | <ul><b>Procedure: </b> Using a ligation calculator the concentration needed was 31.87 ng/ul in a 20ul total ligation. 1ul of PGEX at 105 ng/ul and 6 ul of hCG, 2 ul of T4 buffer, 1 ul of T4 ligase and 10 ul of nuclease-free water. A negative control was used which included digested mamba in pgex with no ligation buffer. The ligation reaction sat at room temperature for 10 minutes. </ | + | <ul><b>Procedure: </b> Using a ligation calculator the concentration needed was 31.87 ng/ul in a 20ul total ligation. 1ul of PGEX at 105 ng/ul and 6 ul of hCG, 2 ul of T4 buffer, 1 ul of T4 ligase and 10 ul of nuclease-free water. A negative control was used which included digested mamba in pgex with no ligation buffer. The ligation reaction sat at room temperature for 10 minutes.</ul> |
− | < | + | </ul> |
+ | <ul> Transformation | ||
<ul><b>Objective:</b>To transform the ligation of hCG and PSB1C3</ul> | <ul><b>Objective:</b>To transform the ligation of hCG and PSB1C3</ul> | ||
<ul><b>Materials:</b>C2987I NEB competent cells, ligation reaction | <ul><b>Materials:</b>C2987I NEB competent cells, ligation reaction | ||
Line 927: | Line 667: | ||
<ul><b>Equipment:</b>shaker and incubator</ul> | <ul><b>Equipment:</b>shaker and incubator</ul> | ||
<ul><b>Procedure:</b><a href="https://static.igem.org/mediawiki/2017/5/56/T--Georgia_State--2.29transformtation.jpg">See attached procedure.</a></ul> | <ul><b>Procedure:</b><a href="https://static.igem.org/mediawiki/2017/5/56/T--Georgia_State--2.29transformtation.jpg">See attached procedure.</a></ul> | ||
− | <ul><b>Results:</b>There was no growth on the ligation plate nor negative control but there was growth on the positive control. Since there was no growth on the ligation plates that would mean the cells have no resistance to ampicillin and the ligation was inefficient due to the fact the hCG was not purified to eliminate contaminants ike EDTA and salts. </ | + | <ul><b>Results:</b>There was no growth on the ligation plate nor negative control but there was growth on the positive control. Since there was no growth on the ligation plates that would mean the cells have no resistance to ampicillin and the ligation was inefficient due to the fact the hCG was not purified to eliminate contaminants ike EDTA and salts. </ul> |
+ | </ul> | ||
</div> | </div> | ||
<div id="date-8-30"> | <div id="date-8-30"> | ||
− | <h1> | + | <h1>Wednesday, August 30th.</h1> |
+ | <li> Cara Jones</li> | ||
+ | <ul>Protein Expression, mamba in pGEX: <a href="https://static.igem.org/mediawiki/2017/e/e1/T--Georgia_State--8_30_17_cj.pdf"> See attached procedure for instructions.</a> | ||
</div> | </div> | ||
<div id="date-8-31"> | <div id="date-8-31"> | ||
− | <h1> | + | <h1>Thursday, August 31st.</h1> |
− | + | </div> | |
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− | </div> | + | |
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<div id="date-9-1"> | <div id="date-9-1"> | ||
− | <h1> | + | <h1>Friday, September 1st.</h1> |
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<div id="date-9-2"> | <div id="date-9-2"> | ||
− | <h1> | + | <h1>Saturday, September 2nd.</h1> |
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<div id="date-9-3"> | <div id="date-9-3"> | ||
− | <h1> | + | <h1>Sunday, September 3rd</h1> |
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<div id="date-9-4"> | <div id="date-9-4"> | ||
− | <h1> | + | <h1>Monday, September 4th</h1> |
</div> | </div> | ||
<div id="date-9-5"> | <div id="date-9-5"> | ||
− | <h1> | + | <h1>Tuesday, September 5th</h1> |
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<div id="date-9-6"> | <div id="date-9-6"> | ||
− | <h1> | + | <h1>Wednesday, September 6</h1> |
</div> | </div> | ||
<div id="date-9-7"> | <div id="date-9-7"> | ||
− | <h1> | + | <h1>Thursday, September 7</h1> |
</div> | </div> | ||
<div id="date-9-8"> | <div id="date-9-8"> | ||
− | <h1> | + | <h1>Friday, September 8th.</h1> |
+ | <li> Tatenda Tela</li> | ||
+ | <ul>hCG for pGEX pcr</ul> | ||
+ | <ul><b>Results</b>:<table><tr><td><img src="https://static.igem.org/mediawiki/2017/f/fb/T--Georgia_State--091317_PCR_of_HCG-Tatenda.jpeg" alt=""></td></table> | ||
+ | <table><tr><th>lane</th><th>contents</th></tr> | ||
+ | <tr><td>1</td><td>Generuler 1kb plus ladder</td></tr> | ||
+ | <tr><td>2</td><td>hCG insert w/ hCG block 5 fwd&rev primers</td></tr> | ||
+ | <tr><td>3</td><td>hCG insert w/ hCG block 5 fwd&rev primers</td></tr> | ||
+ | <tr><td>4</td><td>hCG insert w/ hCG block 5 fwd&rev primers</td></tr> | ||
+ | <tr><td>5</td><td>hCG insert w/ hCG block 5 fwd&rev primers</td></tr> | ||
+ | <tr><td>6</td><td>hCG insert w/ hCG block 5 fwd&rev primers</td></tr> | ||
+ | <tr><td>7</td><td>hCG insert w/ hCG block 5 fwd&rev primers</td></tr> | ||
+ | <tr><td>8</td><td>hCG insert w/ hCG for pGEX fwd&rev primers</td></tr> | ||
+ | <tr><td>9</td><td>hCG insert w/ hCG for pGEX fwd&rev primers</td></tr> | ||
+ | <tr><td>10</td><td>hCG insert w/ hCG for pGEX fwd&rev primers</td></tr> | ||
+ | <tr><td>11</td><td>hCG insert w/ hCG for pGEX fwd&rev primers</td></tr> | ||
+ | <tr><td>12</td><td>hCG insert w/ hCG for pGEX fwd&rev primers</td></tr> | ||
+ | <tr><td>13</td><td>hCG insert w/ hCG for pGEX fwd&rev primers</td></tr> | ||
+ | <tr><td>14</td><td>Generuler 1kb plus ladder</td></tr> | ||
+ | |||
+ | </table></ul> | ||
</div> | </div> | ||
<div id="date-9-9"> | <div id="date-9-9"> | ||
− | <h1> | + | <h1>Saturday, September 9</h1> |
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</div> | </div> | ||
<div id="date-9-10"> | <div id="date-9-10"> | ||
− | <h1> | + | <h1>Sunday, September 10</h1> |
</div> | </div> | ||
<div id="date-9-11"> | <div id="date-9-11"> | ||
− | <h1> | + | <h1>Monday, September 11</h1> |
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</div> | </div> | ||
<div id="date-9-12"> | <div id="date-9-12"> | ||
− | <h1> | + | <h1>Tuesday, September 12</h1> |
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<div id="date-9-13"> | <div id="date-9-13"> | ||
− | <h1> | + | <h1>Wednesday, September 13</h1> |
− | + | <li>Cara Jones</li> | |
− | + | <ul>Overnight Cultures</ul> | |
− | + | <li> Tatenda Tela</li> | |
− | + | <ul>Restriction digest and heat inactivation of samples from September 8</ul> | |
− | + | ||
− | + | ||
</div> | </div> | ||
<div id="date-9-14"> | <div id="date-9-14"> | ||
− | <h1> | + | <h1>Thursday, September 14th.</h1> |
− | + | <li>Tatenda Tela</li> | |
− | + | <ul>Miniprep of overnight cultures prepped by Cara the day before</ul> | |
− | + | <ul><b>Results</b>:<table class="w3-table"> | |
− | + | <tr> | |
− | + | <th>Sample</th> | |
− | + | <th>Concentration (ng/uL)</th> | |
− | + | <th>A260/A280</th> | |
− | + | </tr> | |
− | + | <tr> | |
− | + | <td>1</td> | |
− | + | <td>25</td><td>2.0</td> | |
− | + | </tr> | |
− | + | <tr> | |
− | + | <td>2</td> | |
− | + | <td>7.5</td><td>2.0</td> | |
− | + | </tr> | |
− | + | <tr> | |
− | + | <td>3</td> | |
+ | <td>9.8</td> | ||
+ | <td>1.7</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>4</td> | ||
+ | <td>8.5</td> | ||
+ | <td>1.8</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>5</td> | ||
+ | <td>9.5</td> | ||
+ | <td>1.7</td> | ||
+ | </tr> | ||
+ | </table></ul> | ||
+ | <br> | ||
+ | <li> Cara Jones</li> | ||
+ | <ul>Interlab: Media Preparation: <a href="https://static.igem.org/mediawiki/2017/3/3d/T--Georgia_State--9_14_17_cj.pdf"> See attached procedure for instructions.</a> | ||
</div> | </div> | ||
+ | |||
<div id="date-9-15"> | <div id="date-9-15"> | ||
− | <h1> | + | <h1>Friday, September 15</h1> |
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<div id="date-9-16"> | <div id="date-9-16"> | ||
− | <h1> | + | <h1>Saturday, September 16th.</h1> |
− | + | <li>Victoria Radcliffe, Aditya Natu</li> | |
− | + | <ul>Midiprep of mamba in pGEX to get pGEx backbone</ul> | |
− | + | <table><tr><th>sample</th><th>concentration (ng/uL)</th></tr> | |
− | + | <tr><td>1</td><td>200</td></tr> | |
− | + | <tr><td>2</td><td>275</td></tr> | |
− | + | </table> | |
</div> | </div> | ||
<div id="date-9-17"> | <div id="date-9-17"> | ||
− | <h1> | + | <h1>Sunday, September 17</h1> |
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</div> | </div> | ||
<div id="date-9-18"> | <div id="date-9-18"> | ||
− | <h1> | + | <h1>Monday, September 18</h1> |
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</div> | </div> | ||
<div id="date-9-19"> | <div id="date-9-19"> | ||
− | <h1> | + | <h1>Tuesday, September 19</h1> |
− | + | <li> Tatenda Tela</li> | |
− | + | <ul> Digest of Midiprep of mamba in pGEX done by Victoria and Aditya using EcoRI and NotI enzymes</ul> | |
− | + | <ul><b>Results</b>:<table><tr><td><img src="https://static.igem.org/mediawiki/2017/9/9c/T--Georgia_State--091917_Restriction_digest_of_hcg_in_pGEX-Tatenda.jpeg" alt=""></td></table> | |
− | + | <table><tr><th>lane</th><th>contents</th></tr> | |
− | + | <tr><td>1</td><td>Generuler 1kb plus ladder</td></tr> | |
− | + | <tr><td>2</td><td>Mamba in pGEX</td></tr> | |
− | + | <tr><td>3</td><td>Mamba in pGEX</td></tr> | |
− | + | <tr><td>4</td><td>Mamba in pGEX cut with EcoRI</td></tr> | |
− | + | <tr><td>5</td><td>Mamba in pGEX cut with EcoRI</td></tr> | |
− | + | <tr><td>6</td><td>empty</td></tr> | |
− | + | <tr><td>7</td><td>Mamba in pGEX cut with EcoRI and NotI</td></tr> | |
− | + | <tr><td>8</td><td>empty</td></tr> | |
− | + | <tr><td>9</td><td>Mamba in pGEX cut with EcoRI and NotI</td></tr> | |
− | + | <tr><td>10</td><td>empty</td></tr> | |
− | + | <tr><td>11</td><td>Mamba in pGEX cut with EcoRI and PstI</td></tr> | |
− | + | <tr><td>12</td><td>Mamba in pGEX cut with EcoRI and PstI</td></tr> | |
− | + | <tr><td>13</td><td>empty</td></tr> | |
− | + | <tr><td>14</td><td>empty</td></tr></table></ul> | |
− | + | <li> Tatenda Tela</li> | |
− | + | <ul> Gel extraction of pGEX backbone (lanes 7 and 9)</ul> | |
− | + | <ul> Ligation of digested pGEX backbone and digested hCG from September 13</ul> | |
− | + | <ul> Transforamtion using 5 alpha competent cells | |
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</div> | </div> | ||
<div id="date-9-20"> | <div id="date-9-20"> | ||
− | <h1> | + | <h1>Wednesday, September 20</h1> |
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</div> | </div> | ||
<div id="date-9-21"> | <div id="date-9-21"> | ||
− | <h1> | + | <h1>Thursday, September 21</h1> |
− | + | <li>Tatenda Tela</li> | |
− | + | <ul>Another digest mamba in pGEX and then a ligation with the hCG insert followed by a transformation into 5 alpha competent cells</ul> | |
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</div> | </div> | ||
<div id="date-9-22"> | <div id="date-9-22"> | ||
− | <h1> | + | <h1>Friday, September 22</h1> |
− | + | <li>Julia Ngo<li> | |
− | + | <ul> PCR of hCG using hCG for pGEX fwd and rev primers</ul> | |
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</div> | </div> | ||
<div id="date-9-23"> | <div id="date-9-23"> | ||
− | <h1> | + | <h1>Saturday, September 23</h1> |
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</div> | </div> | ||
<div id="date-9-24"> | <div id="date-9-24"> | ||
− | <h1> | + | <h1>Sunday, September 24</h1> |
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</div> | </div> | ||
<div id="date-9-25"> | <div id="date-9-25"> | ||
− | <h1> | + | <h1>Monday, September 25</h1> |
− | + | <li>Julia Ngo</li> | |
− | + | <ul>Ran gel of hCG PCR from September 22</ul> | |
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</div> | </div> | ||
<div id="date-9-26"> | <div id="date-9-26"> | ||
− | <h1> | + | <h1>Tuesday, September 26</h1> |
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</div> | </div> | ||
<div id="date-9-27"> | <div id="date-9-27"> | ||
− | <h1> | + | <h1>Wednesday, September 27</h1> |
− | + | </div> | |
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− | </div> | + | |
<div id="date-9-28"> | <div id="date-9-28"> | ||
− | <h1> | + | <h1>Thursday, September 28</h1> |
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</div> | </div> | ||
<div id="date-9-29"> | <div id="date-9-29"> | ||
− | <h1> | + | <h1>Friday, September 29</h1> |
+ | <li>Julia Ngo</li> | ||
+ | <ul>Designing Primers for Tac-GST-Mamba in pSB1C3</ul> | ||
</div> | </div> | ||
<div id="date-9-30"> | <div id="date-9-30"> | ||
− | <h1> | + | <h1>Saturday, September 30</h1> |
</div> | </div> | ||
+ | |||
<!-- October --> | <!-- October --> | ||
<div id="date-10-1"> | <div id="date-10-1"> | ||
− | <h1> | + | <h1>Sunday, October 1st.</h1> |
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</div> | </div> | ||
<div id="date-10-2"> | <div id="date-10-2"> | ||
− | <h1> | + | <h1>Monday, October 2nd.</h1> |
− | + | <li> Cara Jones</li> | |
− | + | <ul>Mamba Mutagenesis: <a href="https://static.igem.org/mediawiki/2017/5/5c/T--Georgia_State--10_2_17_cj.pdf"> See attached procedure for instructions.</a> | |
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</div> | </div> | ||
<div id="date-10-3"> | <div id="date-10-3"> | ||
− | <h1> | + | <h1>Tuesday, October 3rd.</h1> |
− | + | <li>Julia Ngo</li> | |
− | + | <ul>PCR of Tac promoter</ul> | |
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</div> | </div> | ||
<div id="date-10-4"> | <div id="date-10-4"> | ||
− | <h1> | + | <h1>Wednesday, October 4th.</h1> |
+ | <li> Cara Jones</li> | ||
+ | <ul>Diagnostic Restriction Digest, Mutagenesis, Transformation: <a href="https://static.igem.org/mediawiki/2017/3/3e/T--Georgia_State--10_4_17_cj.pdf"> See attached procedure for instructions.</a> | ||
+ | <li>Julia Ngo</li> | ||
+ | <ul> PCR Cleanup</ul> | ||
+ | <ul>Diagnostic Gel of PCR</ul> | ||
</div> | </div> | ||
<div id="date-10-5"> | <div id="date-10-5"> | ||
− | <h1> | + | <h1>Thursday, October 5th.</h1> |
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</div> | </div> | ||
<div id="date-10-6"> | <div id="date-10-6"> | ||
− | <h1> | + | <h1>Friday, October 6th.</h1> |
</div> | </div> | ||
<div id="date-10-7"> | <div id="date-10-7"> | ||
− | <h1> | + | <h1>Saturday, October 7th.</h1> |
</div> | </div> | ||
<div id="date-10-8"> | <div id="date-10-8"> | ||
− | <h1> | + | <h1>Sunday, October 8th.</h1> |
</div> | </div> | ||
<div id="date-10-9"> | <div id="date-10-9"> | ||
− | <h1> | + | <h1>Monday, October 9th.</h1> |
− | + | <li> Cara Jones</li> | |
− | + | <ul>Colony PCR, Digest of PCR: <a href="https://static.igem.org/mediawiki/2017/e/ef/T--Georgia_State--10_9_17_cj.pdf"> See attached procedure for instructions.</a> | |
− | + | <li> Julia Ngo</li> | |
− | + | <ul>Redo of PCR of Tac Promoter</ul> | |
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</div> | </div> | ||
<div id="date-10-10"> | <div id="date-10-10"> | ||
− | <h1> | + | <h1>Tuesday, October 10th.</h1> |
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</div> | </div> | ||
<div id="date-10-11"> | <div id="date-10-11"> | ||
− | <h1> | + | <h1>Wednesday, October 11th.</h1> |
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</div> | </div> | ||
<div id="date-10-12"> | <div id="date-10-12"> | ||
− | <h1> | + | <h1>Thursday, October 12th.</h1> |
− | + | <li>Tatenda Tela</li> | |
− | + | <ul>I transformed HCG in PS1C3 into E.coli today</ul> | |
− | + | <li> Cara Jones</li> | |
− | <li> | + | <ul>PCR Amplification, Restriction Digest: <a href="https://static.igem.org/mediawiki/2017/e/e0/T--Georgia_State--10_12_17_cj.pdf"> See attached procedure for instructions.</a> |
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</div> | </div> | ||
+ | |||
<div id="date-10-13"> | <div id="date-10-13"> | ||
− | <h1> | + | <h1>Friday, October 13th.</h1> |
− | + | <li>Tatenda Tela</li> | |
− | + | <ul>I performed PCR and did overnight cultures of 10 colonies</ul> | |
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</div> | </div> | ||
<div id="date-10-14"> | <div id="date-10-14"> | ||
− | <h1> | + | <h1>Saturday, October 14th.</h1> |
− | + | <li>Eudoxie Bataba</li> | |
− | + | <ul><b>Objective:</b>PCR diagnostic gel, Miniprep and Restriction DIgest</ul> | |
− | + | <ul><b>Materials:</b>QIAGEN miniprep kit, 50% glycerol</ul> | |
− | + | <ul><b>Equipment:</b>Centrifuge, Nanophotometer, Thermocycler</ul> | |
− | + | <ul><b>Procedure:</b><a href="https://2017.igem.org/File:T--Georgia_State--HCG_PSB.jpg">See attached procedure.</a></ul> | |
− | + | <ul><b>Result:</b><a href="https://2017.igem.org/File:T--Georgia_State--_PCR_Oct_14.jpg">See attached PCR results.</a></ul> | |
+ | <ul><b>Result:</b><a href="https://2017.igem.org/File:T--Georgia_State--_Miniprep_Oct_14.jpg">See attached miniprep results.</a></ul> | ||
+ | <ul><b>Result:</b><a href="https://2017.igem.org/File:T--Georgia_State--_RD_Oct_14.jpg">See attached Restriction Digest results.</a></ul> | ||
</div> | </div> | ||
+ | |||
<div id="date-10-15"> | <div id="date-10-15"> | ||
− | <h1> | + | <h1>Sunday, October 15th.</h1> |
− | + | <li>Tatenda Tela</li> | |
− | + | <ul>Restriction digests</ul> | |
− | + | <ul>Ligation</ul> | |
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</div> | </div> | ||
<div id="date-10-16"> | <div id="date-10-16"> | ||
− | + | <h1>Monday,October 16th.</h1> | |
− | + | <li>Eudoxie Bataba</li> | |
− | + | <ul> Transformations</ul> | |
− | + | <li> Cara Jones</li> | |
− | + | <ul>PCR amplification of mamba in pGEX, Digest of amplified mamba: <a href="https://static.igem.org/mediawiki/2017/0/08/T--Georgia_State--10_16_17_cj.pdf"> See attached procedure for instructions.</a> | |
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</div> | </div> | ||
− | |||
<div id="date-10-17"> | <div id="date-10-17"> | ||
− | <h1> | + | <h1>Tuesday, October 17th.</h1> |
− | <ul> | + | <li>Tatenda Tela</li> |
− | + | <ul>Ligation</ul> | |
− | + | </div> | |
− | + | <div id="date-10-18"> | |
− | + | <h1>Wednesday,October 18th.</h1> | |
− | </ul> | + | <li>Tatenda Tela and Cara Jones</li> |
+ | <ul>Digested miniprepped DNA from glycerol stocks</ul> | ||
+ | <ul>Gel extraction of the backbones</ul> | ||
+ | <br> | ||
+ | <li> Cara Jones</li> | ||
+ | <ul>PCR of amplified mamba+prefix and suffix: <a href="https://static.igem.org/mediawiki/2017/c/c8/T--Georgia_State--10_18_17_cj.pdf"> See attached procedure for instructions.</a> | ||
+ | </div> | ||
+ | <div id="date-10-19"> | ||
+ | <h1> Thursday, October 19th </h1> | ||
+ | <li>Tatenda Tela</li> | ||
+ | <ul>Double digests</ul> | ||
+ | <ul>Ligations</ul> | ||
+ | <ul>Transformations</ul> | ||
+ | <br> | ||
+ | <li> Cara Jones</li> | ||
+ | <ul>PCR cleanup of mamba: <a href="https://static.igem.org/mediawiki/2017/6/68/T--Georgia_State--10_19_17_cj.pdf"> See attached procedure for instructions.</a> | ||
+ | </div> | ||
+ | <div id="date-10-20"> | ||
+ | <h1> Friday, October 20th </h1> | ||
+ | <li> Tatenda Tela</li> | ||
+ | <ul>Colony pcr</ul> | ||
+ | <ul><table><tr><td><img src="https://static.igem.org/mediawiki/2017/c/ca/T--Georgia_State--_plates_of_hcg_in_psb_with_colonies_picked.jpeg" alt=""></td><tr><td><img src="https://static.igem.org/mediawiki/2017/c/cb/T--Georgia_State--mamba_in_pSB1C3_plates_with_colonies_picked.jpeg" alt=""></td></tr></table></ul> | ||
+ | </div> | ||
+ | <div id="date-10-21"> | ||
+ | <h1> Saturday, October 21st </h1> | ||
+ | <li>Tatenda Tela and Aditya Natu</li> | ||
+ | <ul>Miniprep colonies</ul> | ||
+ | </div> | ||
+ | <div id="date-10-22"> | ||
+ | <h1>Sunday, October 22nd.</h1> | ||
+ | </div> | ||
+ | <div id="date-10-23"> | ||
+ | <h1>Monday, October 23rd.</h1> | ||
+ | <li>Tatenda Tela and Aditya Natu </li> | ||
+ | <ul>Colony PCR</ul> | ||
+ | <ul> Diagnostic restriction digest of colonies </ul> | ||
+ | <ul> Gel extraction and clean-up</ul> | ||
+ | </div> | ||
+ | <div id="date-10-24"> | ||
+ | <h1>Tuesday, October 24th.</h1> | ||
+ | <li>Tatenda Tela </li> | ||
+ | <ul> Digested three different plasmids for ligation with HCG. All were PSB1C3 with different inserts.</ul> | ||
+ | <ul> Gel extraction and clean-up</ul> | ||
+ | </div> | ||
+ | <div id="date-10-25"> | ||
+ | <h1>Wednesday, October 25th.</h1> | ||
+ | <li> Tatenda Tela> | ||
+ | <ul> Minipreps of colonies from the plates from 10/19</ul> | ||
+ | <ul> Restriction digests</ul> | ||
+ | </div> | ||
+ | <div id="date-10-26"> | ||
+ | <h1>Thursday, October 26th.</h1> | ||
+ | <li> Tatenda Tela</li> | ||
+ | <ul> Diagnostic gel </ul> | ||
+ | <ul> Minipreps </ul> | ||
+ | </div> | ||
+ | <div id="date-10-27"> | ||
+ | <h1>Friday, October 27th.</h1> | ||
+ | <strong> DNA Submission Deadline!!</strong> | ||
+ | </div> | ||
+ | <div id="date-10-28"> | ||
+ | <h1>Saturday, October 28th.</h1> | ||
+ | </div> | ||
+ | <div id="date-10-29"> | ||
+ | <h1>Sunday, October 29th.</h1> | ||
+ | </div> | ||
+ | <div id="date-10-30"> | ||
+ | <h1>Monday, October 30th.</h1> | ||
+ | <li> Everyone </li> | ||
+ | <ul><strong>Freak out about our wiki.</strong></ul> | ||
+ | <br> | ||
+ | <li> Cara Jones</li> | ||
+ | <ul>Mamba in pGEX concentration, mamba in pGEX column chromatography, HRV cleavage of mamba-gst fusion, SDS PAGE: <a href="https://static.igem.org/mediawiki/2017/5/5c/T--Georgia_State--10_30_31_17_cj.pdf"> See attached procedure for instructions.</a> | ||
+ | </div> | ||
+ | |||
+ | <div id="date-10-31"> | ||
+ | <h1>Tuesday, October 31st.</h1> | ||
+ | <li> Everyone </li> | ||
+ | <ul><strong>Freak out about our wiki.</strong></ul> | ||
+ | </div> | ||
+ | |||
+ | <div id="date-11-01"> | ||
+ | <h1>Wednesday, November 1st.</h1> | ||
+ | <strong> WIKI FREEZE!!!!!!</strong> | ||
+ | <li> Everyone</li> | ||
+ | <ul><strong>Freak out about our wiki.</strong></ul> | ||
</div> | </div> | ||
Latest revision as of 03:58, 2 November 2017
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Team:Tec-Monterrey/ITESM14 notebook data.html - 2014.igem.org
Cara Jones
Cara Jones
Tori Radcliff
Cara Jones
Cara Jones
Cara Jones
Cara Jones
Cara Jones
Tori Radcliff
Tori Radcliff
Monday, May 1st.
Sunday, June 1st.
Monday, June 2nd.
Tuesday, June 3rd.
Wednesday, June 5th.
Thursday, June 5th.
Friday, June 6th.
Saturday, June 7th.
Sunday, June 8th.
Monday, June 9th.
Tuesday, June 10th.
Wednesday, June 11th.
Thursday, June 12th.
Friday, June 13th.
Saturday, June 14th.
Sunday, June 15th.
Monday, June 16th.
Tuesday, June 17th.
Wednesday, June 18th.
Thursday, June 19th.
Friday, June 20th.
Saturday, June 21st.
Sunday, June 22nd.
Monday, June 23rd.
Tuesday, June 24th.
Wednesday, June 25nd.
Thursday, June 26th.
Friday, June 27th.
- Plasmid Resuspension, Primer Dilutions, PCR: See attached procedure for buffer recipes and instructions.
Saturday, June 28nd.
Sunday, June 29nd.
Monday, June 30nd.
Tuesday, July 1st.
Wednesday, July 2nd.
Thursday, July 3rd.
Friday, July 4th.
Wednesday, July 5th.
Thursday, July 6th.
- Ligation of Mamba insert+pGEX vector and DNA clean up of pGEX+mamba restriction digest: See attached procedure for instructions.
Friday, July 7th.
- Objective:To determine if Alkaline Phosphotase will bind to nitrocellulose paper and produce a signal when mixed with BCIP.
- Materials:Cut small strips of nitrocellulose paper (Nitran), Promega Alkaline Phosphatase (Conc: 1ng/ul), Alkaline 10X buffer diluted, BCIP
- Equipment:UV-Crosslinker
- Procedure:See attached procedure.
- Results: The Ap spread outward but stayed confined meaning the crosslinking worked. Adding the BCIP solution to the tube with the paper produced a noticeable discoloration. Adding the enzyme to the BCIP only produced a small discoloration. The binding of the alkaline phosphatase and BCIP to the paper was successful. The reaction worked only in the intended areas meaning that if the AP is bound to the factor c it may produce a signal only if it is cut, the AP nor BCIP disperses in the solution after being bound to the paper.
![](https://static.igem.org/mediawiki/2017/c/c5/T--Georgia_State--APTest.png)
- Colony PCR: See attached procedure for instructions.
Saturday, July 8th.
Sunday, July 9th.
Monday, July 10th.
- Restriction digest of mamba in pGEX: See attached procedure for instructions.
Wednesday, July 12th.
Thursday, July 13th.
- Transformation of mamba in pGEX into Bl21 Competent cells: See attached procedure for instructions.
Friday, July 14th.
Saturday, July 15th.
Sunday, July 16th.
Monday, July 17th.
- Interlab 2017 transformations and Mamba in pGEX protein expression: See attached procedure for instructions.
Tuesday, July 18th.
Wednesday, July 19th.
Thursday, July 20th.
Friday, July 21st.
Saturday, July 22nd.
Sunday, July 23rd.
Monday, July 24th.
Tuesday, July 25th.
Wednesday, July 26th.
Thursday, July 27th.
- Dilution of Mamba biobrick prefix/suffix, PCR of mamba in pGEX, PCR purification: See attached procedure for instructions.
Friday, July 28th.
Saturday, July 29th.
Sunday, July 30th.
Monday, July 31st.
Tuesday,August 1st.
- Objective:Before the fusion step, the hCG needs to be placed into the PSB1C3 plasmid, so that the hCG can be submitted. In order to add the hCG to add to the plasmid restriction sites need to be added this was done by adding the prefix and suffix through PCR.
- Materials:Qiagen Master mix (2x), Prefix Primer, Suffix Primer, hCG DNA (diluted to 10ng/ul), and Nuclease free H20
- Equipment:Thermocycler
- Procedure:See attached procedure.
- Results:
![]() | ![]() |
Simulated gel using SnapGene | 1% Agarose gel, experimental results |
Wednesday, August 2nd.
Thursday, August 3rd.
- Objective:To double digest both the PSB1C3 backbone and our hCG PCR product
- Materials:DNA of ECFP-PCB1C3 (102 ng/ul) and hCG PCR product, Restriction enzymes: NotI, EcoRI, XbaI, PstI,SpeI, Green Fastdigest Buffer (10x), and Nuclease free H20
- Equipment:Thermocycler
- Procedure:See attached procedure.
- Results:
- 1. 1Kb New England Biolabs Ladder
- 2. hCG uncut
- 3. hCG cut with EcoR1 and Pst1
- 4. hCG cut with Xba1 and Spe1
- 5. ECFP cut with EcoR1 and Pst1
- 6. ECFP cut with Xba1 and Spe1
![]() | ![]() |
Simulated gel using SnapGene | The Wells Contain the following:
|
Friday August 4th.
- Objective: Clean used GST resin so that it may be used in future protein purifications.
- Materials: GST Resin, 70% Ethanol, GST Elution Buffer
- Equipment: Centrifuge,Vortexer, Micropipette,Eppendorf tubes/Falcon tubes, Spectrophotometer, and Cuvettes
- Procedure:See attached procedure for buffer recipes and instructions.
- Results:
Sample | Absorbance @ 280 nm |
---|---|
Clean GST Elution Buffer (Blank) | 0 |
Elution 1 | 0.667 |
Elution 2 | 0.036 |
Saturday, August 5th.
- Objective:To repeat the double digest of PSCB1C3 and hCG PCR
- Materials:DNA of Lt10-PCB1C3 (80 ng/ul) and hCG PCR product, Restriction enzymes: NotI, EcoRI, XbaI, PstI,SpeI, Green Fastdigest Buffer (10x), and Nuclease free H20
- Equipment:Thermocycler
- Procedure:See attached procedure.
- Results:
![]() | Since the diagnostic gel was a success the PSB1C backbone was extracted from the Lt 10 plasmid using an E-gel. |
Sunday, August 6th.
Monday, August 7th.
- Objective:Culture of transformed E.coli with mamba in PGEX
- I picked 10 colonies from plates and did overnight cultures in LB
Tuesday, August 8th.
- Objective:I will get the mamba in PGEX plasmid from transformed E.coli
- Materials:NEB miniprep kit, 50% glycerol
- Equipment:Centrifuge and Nanophotometer
- Procedure:See attached procedure.
- Results:
Sample | Concentration (ng/uL) |
---|---|
1 | 118 |
2 | 123 |
3 | 85 |
4 | 133 |
5 | 90 |
6 | 75 |
7 | 72 |
8 | 100 |
9 | 65 |
10 | 140 |
- SDS PAGE and Coomassie gel stain of purified mamba in pGEX: See attached procedure for instructions.
Wednesday, August 9th.
- Ligation of hCG to PSB1C3
Thursday, August 10th.
- Transformation of hCG-PSB1C3 ligation and overnight culture
Friday, August 11th.
- Colony PCR of hCG-PSB1C3 colonies
Saturday, August 12th.
Sunday, August 13th.
- Overnight culture of hCG-PSB1C3 colonies
Monday, August 14th.
- Miniprep of hCG-PSB1C3 cultures
- Diagnostic restriction digest
- PCR of factor-C gBlocks
- Cara Jones
- Objective: Factor C was divided into 4 G-blocks and ordered from IDT. A PCR was performed to add overlaps to each block so that subsequent overlap extension PCRs could be performed.
- Procedure: See attached protocol.
- Results:
![]() |
A simulated gel was created and then the actual sampled of each were run on an e-gel. 5ul of product with 1ul of 6X dye and 14 ul of DI water was added to each well. The wells include the following from left to right: Gene Ruler 1kb DNA ladder, PCR of g-block 1, PCR of g-block 2, PCR of g-block 3, PCR of g-block 4. |
- Re-do Restrction Digest of mamba: See attached procedure for instructions.
Tuesday, August 15th.
- Overlap Extension PCR
- Objective:To combine the four PCR-ed g-blocks of factor-C.
- Procedure: See attached protocol for details
- Results:
![]() | ![]() |
Simulated gel | Wells contain the following: Gene Ruler 1kb plus MW ladder, Reaction of blocks 1 and 2, Negative control (blocks 1 and 2), Reaction of blocks 3 and 4, Negative control (blocks 3 and 4), Reaction of 1 and 2 with 3 and 4 products, negative control (reactions 1 and 2 with 3 and 3 and 4), and Gene Ruler 1kb plus MW ladder. |
Wednesday, August 16th.
Thursday, August 17th.
- Objective:Prepare hCG for ligation into pGEX through PCR
- Materials:hCG DNA, primers forward and reverse and master mix
- Equipment:Thermocycler
- Procedure:Two 50 ul PCR reactions were performed. 25ul of qiagen mastermix, 2ul of block 5 DNA, 2.5ul of forward primer, 2.5 ul of reverse primer and 18 ul of nuclease-free water was added to tube 1 and tube 2.
- Overnight cultures
- Colony PCR of Cara's transformants
Friday, August 18th.
Saturday, August 19th.
Sunday, August 20th.
Monday, August 21st.
- Miniprep
- Ligations
Tuesday, August 22nd.
Wednesday, August 23rd.
- Diagnostic digest and electrophoresis of mamba: See attached procedure for instructions.
Thursday, August 24th.
- Site directed mutagenesis of mambalgin: See attached procedure for instructions.
Friday, August 25th.
- Objective:To repeat the double digest of PSCB1C3 and hCG PCR
- Materials:DNA of Lt10-PCB1C3 (80 ng/ul) and hCG PCR product, Restriction enzymes: NotI, EcoRI, XbaI, PstI,SpeI, Green Fastdigest Buffer (10x), and Nuclease free H20
- Equipment:Thermocycler
- Procedure:See attached procedure.
- Diagnostic gel of mutanagized mamba: See attached procedure for instructions.
Saturday, August 26th.
Sunday, August 27th.
Monday, August 28th.
- Transformation of Mutagenesis RxNs: See attached procedure for instructions.
Tuesday, August 29th.
- Ligation of hCG and PSB1C3
- Objective:To ligate hCG and PSB1C3
- Materials: digested vector cleaned by Cara Jones, T4 ligase and buffer, digested hCG
- Procedure: Using a ligation calculator the concentration needed was 31.87 ng/ul in a 20ul total ligation. 1ul of PGEX at 105 ng/ul and 6 ul of hCG, 2 ul of T4 buffer, 1 ul of T4 ligase and 10 ul of nuclease-free water. A negative control was used which included digested mamba in pgex with no ligation buffer. The ligation reaction sat at room temperature for 10 minutes.
- Transformation
- Objective:To transform the ligation of hCG and PSB1C3
- Materials:C2987I NEB competent cells, ligation reaction
- Equipment:shaker and incubator
- Procedure:See attached procedure.
- Results:There was no growth on the ligation plate nor negative control but there was growth on the positive control. Since there was no growth on the ligation plates that would mean the cells have no resistance to ampicillin and the ligation was inefficient due to the fact the hCG was not purified to eliminate contaminants ike EDTA and salts.
Wednesday, August 30th.
- Protein Expression, mamba in pGEX: See attached procedure for instructions.
Thursday, August 31st.
Friday, September 1st.
Saturday, September 2nd.
Sunday, September 3rd
Monday, September 4th
Tuesday, September 5th
Wednesday, September 6
Thursday, September 7
Friday, September 8th.
- hCG for pGEX pcr
- Results:
![]() |
lane | contents |
---|---|
1 | Generuler 1kb plus ladder |
2 | hCG insert w/ hCG block 5 fwd&rev primers |
3 | hCG insert w/ hCG block 5 fwd&rev primers |
4 | hCG insert w/ hCG block 5 fwd&rev primers |
5 | hCG insert w/ hCG block 5 fwd&rev primers |
6 | hCG insert w/ hCG block 5 fwd&rev primers |
7 | hCG insert w/ hCG block 5 fwd&rev primers |
8 | hCG insert w/ hCG for pGEX fwd&rev primers |
9 | hCG insert w/ hCG for pGEX fwd&rev primers |
10 | hCG insert w/ hCG for pGEX fwd&rev primers |
11 | hCG insert w/ hCG for pGEX fwd&rev primers |
12 | hCG insert w/ hCG for pGEX fwd&rev primers |
13 | hCG insert w/ hCG for pGEX fwd&rev primers |
14 | Generuler 1kb plus ladder |
Saturday, September 9
Sunday, September 10
Monday, September 11
Tuesday, September 12
Wednesday, September 13
- Overnight Cultures
- Restriction digest and heat inactivation of samples from September 8
Thursday, September 14th.
- Miniprep of overnight cultures prepped by Cara the day before
- Results:
Sample | Concentration (ng/uL) | A260/A280 |
---|---|---|
1 | 25 | 2.0 |
2 | 7.5 | 2.0 |
3 | 9.8 | 1.7 |
4 | 8.5 | 1.8 |
5 | 9.5 | 1.7 |
- Interlab: Media Preparation: See attached procedure for instructions.
Friday, September 15
Saturday, September 16th.
- Midiprep of mamba in pGEX to get pGEx backbone
sample | concentration (ng/uL) |
---|---|
1 | 200 |
2 | 275 |
Sunday, September 17
Monday, September 18
Tuesday, September 19
- Digest of Midiprep of mamba in pGEX done by Victoria and Aditya using EcoRI and NotI enzymes
- Results:
![]() |
lane | contents |
---|---|
1 | Generuler 1kb plus ladder |
2 | Mamba in pGEX |
3 | Mamba in pGEX |
4 | Mamba in pGEX cut with EcoRI |
5 | Mamba in pGEX cut with EcoRI |
6 | empty |
7 | Mamba in pGEX cut with EcoRI and NotI |
8 | empty |
9 | Mamba in pGEX cut with EcoRI and NotI |
10 | empty |
11 | Mamba in pGEX cut with EcoRI and PstI |
12 | Mamba in pGEX cut with EcoRI and PstI |
13 | empty |
14 | empty |
- Gel extraction of pGEX backbone (lanes 7 and 9)
- Ligation of digested pGEX backbone and digested hCG from September 13
- Transforamtion using 5 alpha competent cells
Wednesday, September 20
Thursday, September 21
- Another digest mamba in pGEX and then a ligation with the hCG insert followed by a transformation into 5 alpha competent cells
Friday, September 22
- PCR of hCG using hCG for pGEX fwd and rev primers
Saturday, September 23
Sunday, September 24
Monday, September 25
- Ran gel of hCG PCR from September 22
Tuesday, September 26
Wednesday, September 27
Thursday, September 28
Friday, September 29
- Designing Primers for Tac-GST-Mamba in pSB1C3
Saturday, September 30
Sunday, October 1st.
Tuesday, October 3rd.
- PCR of Tac promoter
Wednesday, October 4th.
- Diagnostic Restriction Digest, Mutagenesis, Transformation: See attached procedure for instructions.
- Julia Ngo
- PCR Cleanup
- Diagnostic Gel of PCR
Thursday, October 5th.
Friday, October 6th.
Saturday, October 7th.
Sunday, October 8th.
Monday, October 9th.
- Colony PCR, Digest of PCR: See attached procedure for instructions.
- Julia Ngo
- Redo of PCR of Tac Promoter
Tuesday, October 10th.
Wednesday, October 11th.
Thursday, October 12th.
- I transformed HCG in PS1C3 into E.coli today
- PCR Amplification, Restriction Digest: See attached procedure for instructions.
Friday, October 13th.
- I performed PCR and did overnight cultures of 10 colonies
Saturday, October 14th.
- Objective:PCR diagnostic gel, Miniprep and Restriction DIgest
- Materials:QIAGEN miniprep kit, 50% glycerol
- Equipment:Centrifuge, Nanophotometer, Thermocycler
- Procedure:See attached procedure.
- Result:See attached PCR results.
Sunday, October 15th.
- Restriction digests
- Ligation
Monday,October 16th.
- Transformations
- PCR amplification of mamba in pGEX, Digest of amplified mamba: See attached procedure for instructions.
Tuesday, October 17th.
- Ligation
Wednesday,October 18th.
- Digested miniprepped DNA from glycerol stocks
- Gel extraction of the backbones
- PCR of amplified mamba+prefix and suffix: See attached procedure for instructions.
Thursday, October 19th
- Double digests
- Ligations
- Transformations
- PCR cleanup of mamba: See attached procedure for instructions.
Friday, October 20th
- Colony pcr
![]() |
![]() |
Saturday, October 21st
- Miniprep colonies
Sunday, October 22nd.
Monday, October 23rd.
- Colony PCR
- Diagnostic restriction digest of colonies
- Gel extraction and clean-up
Tuesday, October 24th.
- Digested three different plasmids for ligation with HCG. All were PSB1C3 with different inserts.
- Gel extraction and clean-up
Wednesday, October 25th.
- Minipreps of colonies from the plates from 10/19
- Restriction digests
Thursday, October 26th.
- Diagnostic gel
- Minipreps
Friday, October 27th.
DNA Submission Deadline!!Saturday, October 28th.
Sunday, October 29th.
Monday, October 30th.
- Freak out about our wiki.
- Mamba in pGEX concentration, mamba in pGEX column chromatography, HRV cleavage of mamba-gst fusion, SDS PAGE: See attached procedure for instructions.
Tuesday, October 31st.
- Freak out about our wiki.
Wednesday, November 1st.
WIKI FREEZE!!!!!!- Freak out about our wiki.