smori at nmsu.edu
Fri Dec 23 19:20:03 EST 1994
Reid, Thomas M. (tar9 at NIOBBS1.EM.CDC.GOV) wrote:
: I am attempting to use RT-PCR to amplify the cDNA for the human hprt gene.
: The source of the RNA is a human lymphoblast cell line. When I use cells
: actively growing in culture I am usually successful. In fact as few as 100
: cells works fine. The problem occurs when trying to amplify using frozen
: cells. The cells ( 10^6 to 10^7) are frozen in a volume of 0.5 ml
: containing tissue culture medium and DMSO. I have tried using
: phenol-chloroform extraction as well as commercial phenol-guanidinium
: thiocyanate solutions to prepare total RNA. I use at least 3 ug of RNA
: along with a gene specific primer for the RT reaction. The RT reaction is
: diluted 1:5 and 5 ul used for PCR in a total reaction volume of 50 ul. PCR
: requires 2 rounds of amplification - the second using nested primers. In
: the majority of cases I get no PCR product.
I am gonna suggest here that it is not the PCR portion that is giving you
problems. I think it is the RT reaction. Remember that DMSO at amounts
equalling to 17% V/V inhibits RT. Formamide at 5% V/V. 30mMGuanidine-HCL,
30mM GITC and 1mM EDTA.
For more detail look up Focus 16 (3): 89, (4): 102.
Shahram Mori _/\_
Program in Molecular Biology _\ /_ Saskatoon/SK/CANADA
Dept. of chemistry and Biochemistry Box 3C \_ _/
NMSU Las Cruces NM ||
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