PCR on whole yeast cells

Dr. I. Dunham idunham at crc.ac.uk
Thu Jun 9 12:52:06 EST 1994


In article <A.C.Hilton-080694094124 at bcs88.bham.ac.uk> you write:
>In article <2sumhu$spf at mercury.dur.ac.uk>, Bert Popping
><Bert.Popping at durham.ac.uk> wrote:
>
>> Dear Alvin,
>> 
>> I haven't found any reference in a journal, but there is a summary of this 
>> topic in the archive. I used this technique last week and I got excellent 
>> results! The reason why you can't find it in the literature is propbably 
>> because it's just to simple. 
>> 

Back in 1990 Eric Green and I tried a number of ways to make PCRable
DNA from yeast cells for analysis of YACs.  After various minimalist 
procedures we found that anything we did to the cells actually made the 
situation worse, and just PCR'ing from a tiny bit of the colony picked
off with a toothpick was the best solution.  It is also possible to use
a larger amount of colony (about the amount you can get on the fat end
of the toothpick) resuspended in TE (0.1mM EDTA), and use this suspension
many times - we have YAC colony suspensions that are still reproducibly
amplifiable by AluPCR a year later.  We did publish this but perversely we
dressed it up in a method to determine yeast mating type by PCR

Huxley, Green and Dunham (1990) Trends in Genetics 6 236.

Incidently Eric Green has recently found that this colony PCR does not work 
reproducibly in thermocylcers with a heated top plate if you omit the 
mineral oil.

Ian
===========================================================================

Ian Dunham 
Sanger Centre
Hinxton Hall
Hinxton
Cambridge
CB10 1RQ

id1 at sanger.ac.uk
===========================================================================

>> But here comes the summary:
>> 
>> 
>> Let me summarize our current experience:
>> 
>> 1.  As many of you have said, THERE IS NO NEED TO ISOLATE DNA.  We have  
>> found that one toothpick's worth (we use the small end of a disposable  
>> innoculating loop to avoid contamination), or about 1/2 of a 2 day YPD  
>> colony, resuspended directly in a 100 microliter PCR reaction, works just  
>> fine.  There is also no need to preboil; just resuspend and go.  
>> 
>> 2.  We have also found that the PCR product is cuttable by at least one  
>> restriction enzyme (Sal1) DIRECTLY from the tube.  Kind of surprising with  
>> all those yeast guts swimming around, but there you have it.  Just spin the  >> tube to pellet the glop and use the supernatant.
>> 



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