Team:EPF Lausanne/Notebook/Yeast
saccharomyces cerevisiae
pTPGI_dCas9_VP64 integration
As we need dCas9 expressing yeasts, we decided to integrate a plasmid that would inducively express dCas9.
First and second trial
The negative and positive control worked fine, but the transformation control didn’t work. After investigation we found out that we didn’t have an origin of replication in our plasmid thus not enabling our yeasts to replicate the plasmid and to grow. See more details here
Assemble pdCas9-wOpen pdCas9 by PCR
We received plasmid pdCas9 in bacteria and did a Miniprep (cf. Protocols) on overnight cultures to isolate it. This step consists of linearizing pdCas9 by PCR.
Materials and method
- 20 µl Phusion PCR (cf. Protocols) of pdCas9 with primers f_Gbs_pdCas9 and r_Gbs_pdCas9
- PCR product purification (cf. Protocols)
- Agarose gel electrophoresis of purified PCR products
Results
Linearized pdCas9-w is expected to be 6705 bp.The first try of this PCR was unsuccessful (gel not shown here). For our second try, we tested many parameters: HF vs. GC buffer, different annealing temperatures and different extension times. This time, many, but not all, of our samples were successfully amplified (cf. figure 1). The difficulty of this PCR is probably due to the fact that the size of the ampicon is very long, almost 7 kb.For next steps, sample from lane 1 (cf. figure 1) was used.
