PCR from few cells.

Klaus.Matthaei at anu.edu.au Klaus.Matthaei at anu.edu.au
Wed Aug 26 17:13:54 EST 1992


>Hello.
>Does anyone out there have a 
>protocol for using the PCR on the DNA from
>a small number of cell; 10-1000 mamalian cells?
>Ideally this will be able to work from tissue culture cells 
>being cloned by limiting dilutions in 96 well
>flat-bottomed tissue culture plates.  Thanks in advance.
>
>Mark Brudnak
>University of Tulsa
>INTERNET: BMA14432 at VAX1.UTULSA.EDU

G'Day
Some years ago we developed a PCR method for sexing pre-implantation cattle
embryos during commercial embryo transfer.  This was usually done on the
kitchen table in the farmhouse so the method had to be simple and
foolproof.  If your numbers of cells is not too high (< 500-1000), wash
your cells and resuspend in 10-20  l of water.  Boil 10 minutes and add the
rest of your PCR cocktail  (we used to make a mix containing  everthing
including the taq and freeze in Liquid nitrogen) to say 50  l, add oil and
cycle.  We are able to accurately sex a single cell this way (~1000 target
molecules since it was a repeat) which we obtain by biopsy of the embryo at
the blastocust or morula stage. 

Hope this helps.

Cheers, Klaus.
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Klaus Matthaei
Gene Targeting
The John Curtin School of Medical Research
The Australian National University
E-mail: Klaus.Matthaei at anu.edu.au

"Don't try and fix it if it ain't broke".
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