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 in advance!
Maybe you should described what you try to do and at which steps do you have trouble. Maybe joining few documents like gel picture would help to tailored some helping hand
I have a very similar problem, rescuing a (bigger) plasmid of a maxiprep, hope someone can help us
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