PCR out of 20 cells

Wolfgang Wybranietz wolfgang.wybranietz at uni-tuebingen.de
Wed Sep 3 12:04:08 EST 1997


I am desperately looking for a protocol on how to PCR-amplify a sequence
out of (stably integrated, human) genomic DNA from not more than 50
cells.
My selection method yields neomycin resistant clones of no more than 20
to 50 cells. 
Is there a method to either effectively extract the genomic DNA out of
so little cells for later use in PCR 
or to directly lyse the cells in the cycler and start the PCR in the
same tube?
There are some protocols in the net for isolation of chromosomal DNA
from "small numbers of mammalian cells", but in all protocols downloaded
so far it turned out that "small numbers" in these cases meant 10E4 to
10E5 cells!
What I am dealing with is really not more than 20 cells. 
I also know that for "single cell RT-PCR" the single cell is sucked into
the PCR tube right away, 
but is there a working protocol for genomic DNA?

Wolfgang Wybranietz



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