RT-PCR problems

Mark Fry 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?  

Please help! 



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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