Summary-PCR few cells

BMA14432 at VAX1.UTULSA.EDU BMA14432 at VAX1.UTULSA.EDU
Mon Aug 31 09:29:38 EST 1992


Well, it looks like all the votes are in so here it is.
It seems most of the protocols are pretty similar.
They all follow the basic spin down cells, wash in buffer of choice(PBS,TBS ect)then lyse cells. How they go about this is somewhat varied. One suggestion is to dry cells then all a 5x mix(1 MTris-Cl 8.5
250 ul 1MKCl 188 ul, 1 M MgCl2 15 ul, 1 M DTT 50 ul, 2.9 ug/ul BSA 125 ul
NP-40(your detergent) 125 ul, H2O to 1000 ul total. Incubate 37 C 10 min.
 then add 5 ul of 10x PCR Buffer, 1 ul each primer(O.D.=0.5)
2 ul 10 mM dNTPs, 0.5 ul Taq(2.5u)
35.5 ul H20. then cycle.  This does not call for any Proteinase K which 
makes the protocol more attractive in my opinion. Why? Well, I have
found that  even after inactivation the proteinase K still inhibits the 
PCR process(granted not to a large degree but if your going after a 
single copy gene form just a few cells it becomes a big deal.)
Another protocol calls for Proteinase K and SDS as the detergent. 
Here again I prefer not to use SDS because it seems to have an negative
effect on the PCR.  There is a protocol in PCR Protocols-A Guide to 
Methods and Applications. edited by Innis, Gelfand, Sninsky, and White
which I have used successfully however it seems to come up short
in my hands when the cell number drops lower (below 100 cells).

Hope this is enough of a summary, but if not feels free to contact me
via e-mail.

Mark Brudnak
University of Tulsa
INTERNET: BMA14432 at VAX1.UTULSA.EDU



More information about the Methods mailing list