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

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.

Materials and method

• 20 µl Phusion PCR (cf. Protocols) with 1 ng pWJ66 using HF buffer and following thermocycling settings:

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

• PCR product purification (cf. Protocols)
• Agarose gel electrophoresis of purified PCR products

Results