Protocol for PCR straight from yeast colony?

Georg Lipps Georg.Lipps at biochemie.med.uni-giessen
Sun Apr 9 06:47:36 EST 1995


In article <susans-010495113900 at triezenberg2.bch.msu.edu>, susans at student.msu.edu (Susan M. Sullivan) says:
>
>I need to verify if the plasmids that I transformed into my S. cerevisae
>are correct.  I know that it is possible to pick a colony of yeast, lyse
>it, and PCR amplify a fragment of DNA using the DNA retrieved from the
>yeast colony as the template.  However, I do not have a protocol for this
>procedure.  If anyone knows a protocol or where to find it please let me
>know. 
>
>Thanks
>
>John Stebbins

I tested my transformation of S.c. the following way:

Lyse yeast cells with glass beads (heating does not work) and add 1µl of
the supernatent to 9µl of a PCR mixture.  Temperature depends on your primers, 
a good first try is  94° C, 55° C and 72° C for 45 seconds each and 25 cycles.
My starting material were usually minimal media grown liquid cultures, but resuspension
of a colony in water should also work given the little amount of cells needed.  It always worked,
a nice control is to add the plasmid in a reaction with untransformed yeast cells.

Good Luck,

Georg Lipps    



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