Difference between revisions of "Team:Bielefeld-CeBiTec/Notebook/HeavyMetals"
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<li>Electroporation resulted in colonies, which were screened with a colony PCR.</li> | <li>Electroporation resulted in colonies, which were screened with a colony PCR.</li> | ||
<li>A product length of 1705 bp was expected. The gel shows that most plasmids seem to be correct.</li> | <li>A product length of 1705 bp was expected. The gel shows that most plasmids seem to be correct.</li> | ||
| + | <li>This result was confirmed by sequencing.</li> | ||
</ul> | </ul> | ||
</ul> | </ul> | ||
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<div id="collapseEight" class="panel-collapse collapse"> | <div id="collapseEight" class="panel-collapse collapse"> | ||
<div class="panel-body"> | <div class="panel-body"> | ||
| − | < | + | <ul> |
| + | <li>Insertion of a second repressor binding site into BBa_J33201</li> | ||
| + | <ul> | ||
| + | <li>Two primer pairs were used to amplify the plasmid and introduce a second repressor binding site after arsR</li> | ||
| + | <li>A PCR with Phusion Polymerase failed. A gradient PCR did not show any improvement.</li> | ||
| + | <li>A comparison of Phusion and Q5 showed that Q5 works very well.</li> | ||
| + | <li>However, an upscalling of the reaction to produce more product did not work.</li> | ||
| + | <li>Therefore, two 20 µL reactions were used per fragment. This PCR worked well and the products were extracted from the gel.</li> | ||
| + | <li>We performed a Gibson assembly reaction with the two fragments and transformed the mix via heat shock.</li> | ||
| + | <li>A colony PCR was performed with five colonies, but only one reaction yielded product.</li> | ||
| + | <li>Five colonies were used to inoculate overnight cultures.</li> | ||
| + | <li>Plasmids were isolated from the cultures and a restriction digest was performed to check them.</li> | ||
| + | <li>The bands were as expected for all colonies.</li> | ||
| + | <li>One plasmid was handed in for sequencing, which confirmed its correctness.</li> | ||
| + | </ul> | ||
| + | </ul> | ||
</div> | </div> | ||
</div> | </div> | ||
Revision as of 15:20, 17 June 2015
- We transformed BioBricks from the distribution:
- BBa_J33201 (ArsR)
- BBa_K516030 (mRFP1 with RBS and terminator)
- Colonies were used to inoculate overnight cultures.
- Plasmids were isolated using a miniprep kit.
- ArsR (BBa_J33201) and RFP (BBa_K516030) were combined using 3A assembly:
- Restriction digest of arsR, RFP and pSB1K3.m1
- PCR Clean-up
- Ligation: 2 µL per fragment, 30 min
- Transformation by electroporation and heat shock (for comparison)
- No colonies, therefore the ligation was repeated with significantly more DNA. Incubation over night.
- Electroporation resulted in colonies, which were screened with a colony PCR.
- A product length of 1705 bp was expected. The gel shows that most plasmids seem to be correct.
- This result was confirmed by sequencing.
- Insertion of a second repressor binding site into BBa_J33201
- Two primer pairs were used to amplify the plasmid and introduce a second repressor binding site after arsR
- A PCR with Phusion Polymerase failed. A gradient PCR did not show any improvement.
- A comparison of Phusion and Q5 showed that Q5 works very well.
- However, an upscalling of the reaction to produce more product did not work.
- Therefore, two 20 µL reactions were used per fragment. This PCR worked well and the products were extracted from the gel.
- We performed a Gibson assembly reaction with the two fragments and transformed the mix via heat shock.
- A colony PCR was performed with five colonies, but only one reaction yielded product.
- Five colonies were used to inoculate overnight cultures.
- Plasmids were isolated from the cultures and a restriction digest was performed to check them.
- The bands were as expected for all colonies.
- One plasmid was handed in for sequencing, which confirmed its correctness.