PCR using Yeast colonies-info wanted

Aidan Weatherill 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.


BEGIN-

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 
conditions).

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,
Greg Tomlin
****************
Dept. of Biochemistry,
UMIST, P.O.Box 88,
Manchester, M60 1QD,
U.K.

END-

It is extreamly important to use fresh colonies, and either NEW sterile
toothpicks or sterile pipette tips. 

Aidan.



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