Yeast Colony PCR

Richard J. Reece rreece at fs2.scg.man.ac.uk
Fri Jan 26 03:04:14 EST 1996


Simon Twigger wrote:
> 
> I am using the Clontech 2 hybrid system and its come time to check my
> postitive colonies. The manual suggests doing a quick/crude plasmid prep
> for each clone, then PCRing off that and then restricting the PCR product
> using AluI and/or HaeIII to try and identify similar clones - thereby
> cutting down on the amount of sequencing to be done.
> 
> I was wondering if there is a protocol for Yeast Colony PCR, like the one
> we regularly use with bacteria, so that I can PCR my product directly
> from a colony without having to go through the plasmid step? I have heard
> that the problem tends to be getting the yeast open and also the genomic
> DNA contamination.
> 
> Has anyone tried this/published anything on this anywhere?
> 
> Thanks for any help anyone can give me
> 
> Simon Twigger,
> Medical College of Wisconsin, Milwaukee.

I find that PCR from yeast colonies (either amplifying genomic or 
plasmid DNA sequences) works really well.  The colony (2-3 mm from a 
fresh plate) is picked with a sterile 18G needle directly into a PCR 
tube containing olgios, taq, dNTPs etc.  This is then vortexed 
briefly and placed directly into the thermocycler (I use one with a 
heated lid so there is no need for an oil overlay - if you need oil, 
make sure that you do not microfuge the samples after adding the 
oil).  The main tricks with getting this to work are the freshness 
of the colonies on the plate and the amount scraped onto the needle 
and put into the PCR tube.  Here is one case when less is better.  
You should get the smallest amount of yeast that you can see with 
the naked eye onto the end of the needle.  I tend to just gently 
swipe the surface of the colony with the needle to try to pick up as 
little as possible.

I can usually detect genomic DNAs in 50 cycles and plasmid on 20-30.

Richard Reece,
School of Biological Sciences,
University of Manchester,
Manchester M13 9PT
United Kingdom



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