PCR from yeast plasmid

[Michael Lichten]

>Hi,
>I am trying to do PCR from yeast plasmids and am not having any success.
>I have made 'yeast extract' according to the protocol in the Red Book
>(current protocols in molecular biology), I can transform E. Coli with the
>extract, but for some reason my PCR is not working.  Is there something
>that I am missing?  Any help would be greatly appreciated, Thank You
>Nick Kaplinsky

There is an extensive discussion of how to PCR from yeast colonies in the
archives of this listserve, accesible through the Saccharomyces Genomics
Database Web page (http://genome-www.stanford.edu/).  In brief, we do the
following:

1. Take 1/4 to 1/2 of a freshly grown yeast colony on the end of a clean
toothpick.  Resuspend in a conventional 50 ul PCR reaction.

2.  Do a standard PCR.

In other words, there's no need even to purify your plasmid.


Michael Lichten
lichten at helix.nih.gov