RT-PCR Help Me :)

steven joseph chmura sjchmura at kimbark.uchicago.edu
Fri May 21 12:16:49 EST 1993


I am attempting to use RT-PCR to dectect low levels of mRNA expression.
So far I have created a huge mess.  I have non-specific binding problems
and high bckground noise.  I am using the GIBCO BRL SuperScript kit.

COnditions:

Primers for RT and PCR: 20bp 130 bp apart
PCR conditions :
	94 at 1min
	50 at 2,72 at 2 min
	72 at 7 min

The mRNA was extracted from liver tissue.

I am wondering ther following:

Would RNase free DNase a wise treatemtn before the RT reaction
to minimize genomic DNA contamination?

Make the PRimers longer?

Change the PCR annealing times?

Any help from people who have gotten nice tight bands (like the
ones seen in the manual) would be greatly appreciated.

Thanks,,,....


-- 
________________________________________________________________________________Steven Chmura				University of Chicago Medical School(M1)
  "Given enough time, the impossible becomes probable, and the probable
	inevitable.."  -George Wald, "On the Origins of Life"



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