whole-cell yeast PCR

betina at univ-lyon1.fr betina at univ-lyon1.fr
Mon Sep 29 08:03:00 EST 1997


In article
<Pine.GSO.3.96.970927112920.14038B-100000 at piglet.cc.utexas.edu>, Justinian
<jtbrown at ccwf.cc.utexas.edu> wrote:

>         Has anyone had any success with a whole-cell PCR protocol from
> Saccharomyces or other yeast?  Any leads would be appreciated.
> 
>         thanks,
> 
>         Justin

I'm using the following protocol for Kluyveromyces lactis, that I have
picked somewhere on the Internet. Usually it works fine.

                              Yeast Colony PCR

Stuff you need:

10X Colony PCR Buffer
0.125 M Tris-HCl pH 8.5
0.5625 M KCl

1.  Using pipet tip, add a very small amount of cells
    (~0.25 µl worth) into PCR reaction mix (20 µl reaction).

    Reaction mix (combine on ice):

                  2 µl 10X Colony PCR Buffer
                1.2 µl 25 mM MgCl2
                0.4 µl 10 mM dNTP's
                 10 pmols of each primer
                0.2 µl Taq polymerase
                ____________________________
                ==> water to 20 µl

2.  PCR cycle profile:

                    94 deg C    8 minutes
                    ______________________
                    94 deg C    1 minute
                    55 deg C    45 seconds
                    72 deg C    2 minutes

                    ==> 35 cycles
                    ______________________
                    72 deg C    7 minutes
                     4 deg C    infinity

3.  Load entire sample on agarose gel.

Good luck,

Svato



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