Team:EPF Lausanne/Notebook/Ecoli
E. Coli Laboratory Notebook
Assemble pdCas9-w: Open pdCas9 by PCR
We received plasmid pdCas9 in bacteria. We did a Miniprep (cf. Protocols) on overnight cultures to isolate it.pdCas9 is a plasmid containing dCas9 under a Tetracyclin inducible promoter and a Chloramphenicol resistance gene, available on Addgene under the name pdCas9-bacteria.We opened this plasmid by PCR to be able use later on in a Gibson assembly.
Materials and method
- 20 µl Phusion PCR (cf. Protocols) of 1 ng pdCas9 with appropriate primers, testing parameters such as HF vs. GC buffer, annealing temperatures and extension times
- PCR product purification (cf. Protocols)
- Agarose gel electrophoresis of purified PCR products
Results
Linearized pdCas9-w is expected to be 6705 bp.After testing many parameters, we were able to linearize the plasmid succesfully, as seen on gel above.For further uses, sample from lane 1 was used. It was prepared with HF buffer and the following thermocycling settings:
| Step | Temperature | Time | |
|---|---|---|---|
| Initial Denaturation | 98°C | 40 seconds | |
| 25 cycles | Denaturation | 98°C | 15 seconds |
| Annealing | 59°C | 22 seconds | |
| Extension | 72°C | 2 minutes 30 seconds | |
| Final Extension | 72°C | 7 minutes | |
| Hold | 4°C |
Assemble pdCas9-w: Extract w subunit from pWJ66 by PCR
We received plasmid pWJ66 in bacteria. We did a Miniprep (cf. Protocols) on overnight cultures to isolate it.pWJ66 is a plasmid containing dCas9 fused at its C-terminal to the w subunit of RNA polymerase as well as a tracrRNA gene and a CRISPR array gene, available on Addgene.We extracted the w subunit to fuse it to our own dCas9 by Gibson assembly.
Materials and method
- 20 µl Phusion PCR (cf. Protocols) with 1 ng pWJ66 using appropraite primers, HF buffer and following thermocycling settings:
- PCR product purification (cf. Protocols)
- Agarose gel electrophoresis of purified PCR products
| Step | Temperature | Time | |
|---|---|---|---|
| Initial Denaturation | 98°C | 30 seconds | |
| 30 cycles | Denaturation | 98°C | 10 seconds |
| Annealing | 62°C | 15 seconds | |
| Extension | 72°C | 15 seconds | |
| Final Extension | 72°C | 7 minutes | |
| Hold | 4°C |
Results
Successful PCR reactions are expected to yield 340 bp fragments.As seen on gel, PCR was succesful for sample in lane 1.
Assemble pdCas9-w: Gibson assembly of pdCas9-w
Using PCR products from experiments described above, we fused the w subunit of DNA polymerase to dCas9 by Gibson assembly.
Materials and method
- Gibson assembly (cf. Protocols) with purified PCR products:
- Open pdCas9: 0.02 pmol = 95 ng
- w subunit extracted from pWJ66: 0.06 pmol = 12.5 ng