FW: How to crack yeast for PCR ??

Stewart, Robert rob.stewart at labatt.com
Thu Apr 11 12:50:51 EST 1996


Greetings,
For cracking yeast cells we have employed two methods: one is subjecting the 
cells to several thermal cycles between 4 degrees and 95 degrees, and 
following centrifugation in a microcentrifuge, PCR.  We could detect to a 
limit of about 400 cfu yeast.  We get much better sensitivity using a 
commercially available extraction kit containing fine glass beads which 
takes about 30 minutes.  We haven't used using transformed yeast, but this 
shouldn't make a difference with relatively small PCR amplifications (<2500 
bp or so) I hope this is helpful.
Cheers
RS
----------
From: 
	liss at alf1.ngate.uni-regensburg.de[SMTP:liss at alf1.ngate.uni-regensburg.de]
Sent: 	Wednesday, April 10, 1996 11:32AM
To: 	methods at net.bio.net
Subject: 	How to crack yeast for PCR ??

Hi,

Transformation of yeast is pretty easy...
BUT to my experience yeast is a fairly unstable organism
doing some weird thing like loosing the plasmid and still 
growing on selective dropout medium...
One way to reconfirm a successful transformation is DNA-isolation, 
followed by electroporation of E. coli. Rather time consuming...
So I thought about a quick PCR-test, amplifying the insert in the
yeast-vector. I even found a paper describing such a test 
(Liang & Richardson (1992) Biotechniques, 13(5): 730-2)
However in their paper they still do a DNA-isolation (glassbeads & 
phenol/CHCl3) from yeast cells prior the PRC reaction.

Let me come to my question:
Is this DNA-isolation really necessary or is there an easier way to
perform a reliable PCR. (Just like with E. coli where you just take a 
little colony and add the whole cells to your reaction.)
If anybody has experience with this or can contribute any other 
suggestions on how to run the PCR etc. please let me know...

Thanx in advance.

Mike








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