Make pdCas9-w

Fuse dCas9 (from plasmid pdCas9) with the w subunit of polymerase (from plasmid pWJ66) to allow dCas9 to activate a gene when it binds upstream of the promoter.

We received the plasmids in bacteria. We did a Miniprep (cf. Protocols) on overnight cultures to isolate the plasmids.

We opened pdCas9 and extracted the w subunit from pWJ66 by PCR. We assembled these parts with Gibson assembly.

Open pdCas9 by PCR and extract w subunit from pWJ66 by PCR

05.06.2015

Materials and method

Phusion PCR of 1 ng pdCas9 with HF buffer, annealing temperature (Ta): 58°C, extension time (ext): 150 sec, 30 cycles

Phusion PCR of 1 ng pWJ66 with HF buffer, annealing temperature (Ta): 62°C, extension time (ext): 15 sec, 30 cycles

PCR products were analyzed after PCR Product Purification (cf. Protocols) by 1,2% agarose gel electrophoresis.

Results