PCR using Yeast colonies-info wanted
aweatherill%immads2.jr2 at ox.ac.uk
Tue Nov 26 10:52:42 EST 1996
In article <stebbin1.1199080292A at nntp.msu.edu>, stebbin1 at student.msu.edu
(John Stebbins) wrote:
> I am going to try to pcr a fragment out of yeast genomic DNA. I seem to
> remember a post that descibed adding some yeast straight from a plate into
> the reaction and letting the 95degrees lyse it. Anyone know the details of
> this and how well it works?
> Thanks...John Stebbins
In response to a similar request I was sent the following protocol which
works, though is fairly Taq heavy. It uses a microwave to lyse the cells.
A method that I've used with consistent success is:---
1. Prepare a mixture of 10mM Tris-HCL (pH8), 2mM MgCl2, 50mM KCl, 0.2mM
of each dNTP and 0.2microM of each primer (i.e. fairly standard PCR
2. In a separate PCR Eppendorf, smear cells from a FRESH colony on the
base of the tube.
3. Heat the cells in a microwave on full power for one minute, then
IMMEDIATELY place into ice-water to cool the tube.
4. Take 50microliters of the mix prepared in step 1 and fully resuspend
the microwaved cells in it.
5. Add 5Units of Taq and run under whatever PCR conditions are
appropriate for your primers and length of product (if you wish, you can
overlay the reaction mixture with 25microliters of mineral oil, but when
using a PCR machine with a heated lid I don't find this necessary).
6. I generally run 15-20microliters of this mixture in a gel, although
you could often get away with running only 10microliters.
I've managed to colony PCR fragments up to 4kb with this method. Let me
know if you have any queries or problems with this procedure.
Best of luck,
Dept. of Biochemistry,
UMIST, P.O.Box 88,
Manchester, M60 1QD,
It is extreamly important to use fresh colonies, and either NEW sterile
toothpicks or sterile pipette tips.
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