Hello, I am new to yeast research. I am trying to rescue plasmid from yeast and have tried several protocols developed by researchers. But the results are not promising. I ended up buying this kit (yeast plasmid miniprep I) from zymo research and I still see no colony after the bacterial transformation.
The plasmid: I am trying to insert a 2kb fragment into a 2μ vector cut with PstI and NotI. I have used the primer to create 40bp overlap between the fragment and the vector, and transformed them into yeast for homologous recombination. I got no colonies with the negative control (no fragment), and many colonies on the transformation plate. The gel electrophoresis of colony PCR showed right size bands.
Now I want rescue this plasmid for sequencing by tranforming it into E.coli. The problem is the bacteria transformation did not work so far (as I described earlier). Hope to find some suggestions from people who had been in similar situation!
Thanks to any contributions in advance!
Plasmid rescue from yeast should be pretty straightforward, although it's been some years since I last did it, I don't recall having had much difficulty. Are you sure your competent cells are good and you get excellent results with a control transformation with uncut vector?
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