Yeast are tricky for colony PCR, hard to pop the cells, plasmid isn't high copy. Rather than colony PCR, you could try plasmid extraction and E. coli transformation. Your bait and prey plasmids should have different selectable markers for growth in bacteria, just plate onto the appropriate antibiotic and wait for bacterial colonies. Then, try your colony PCR on the E. coli or just use them for plasmid preps. The extracted plasmid can be used for some diagnostic digests and sequencing.

Zymoprep is a pretty good kit for extracting plasmids from yeast.

Amy Klocko

Thank you for your answer. I understood your recommendation but the problem is sometimes I got around 30 ~ 40 colonies on my quadruple plates. In this case, I couldn't do plasmid rescue for all of them (actually it's possible but it will take a lot of time). That's why I'm doing yeast colony pcr for my first screening after mating. My biggest concerning is that I thought my library got contaminated or have some problems because of I failed in my yeast colony pcr. But, do you think it's not a problem? It may because of some tricky of yeast cells..?

My workflow is like that after mating -> yeast colony pcr -> purification and sequencing -> blast; if the sequence is interesting -> plasmid rescue -> co-transformation in yeast and confirm the interaction.