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.

Materials and method

  • 20 µl Phusion PCR (cf. Protocols) with 1 ng pdCas9 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

After testing many parameters, we were able to amplify the plasmid succesfully, as seen on gel above (Date: 05/10/15).
For further steps 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 will extract the w subunit to fuse it to our own dCas9.

Materials and method

  • 20 µl Phusion PCR (cf. Protocols) with 1 ng pdCas9 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

Still under construction