wmfry at morgan.ucs.mun.ca
Wed Nov 1 16:33:11 EST 1995
I'm having a problem with my RT-PCR reaction. I have done the RT three
times and still no PCR products.
I start with about 2ug total RNA (in 16ul depc H2O) and I know this is
good quality stuff.Heat the RNA to 80 C for 3 min, place on ice for 3 min.
I add (in order, on ice) 1.5 ul dNTP (10 mM each), 1 ul of 100 ng/ul oligo
dT adaptor [primer 34 mer, 17-18 of which are dT), 6 ul 5X buffer (gibco),
3 ul 0.1M DTT (gibco), 2.5 ul of superscript MMLV (Gibco). Incubate at 37
C for 1 hour, and use as template in PCR.
I run the PCR reaction along with a control template of a clone that
gives the right product, so I know the primers work fine.
I have also done this RT-PCR before, and it worked fine.
Is it wise to treat the cDNA with RNase H or RNase A? Is there
anything else I should Do?
Mark Fry ribit @..@
4th Floor BMS, Fac. of Med. \ (----)
Memorial University of NFLD ( >**< )
A1B 3V6 ^^^""""^^^
wmfry at plato.ucs.mun.ca (Xenopus)
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