RT-PCR problems

Lauryl Maiya J Nutter lmjnutte at acs5.acs.ucalgary.ca
Wed Nov 1 21:57:17 EST 1995


Mark Fry (wmfry at morgan.ucs.mun.ca) wrote:

<snip>
: 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)

You might want to try running your RT extension at a higher
temperature (42-50 C).  This will help reduce secondary structure
interference with cDNA extension.  It may be that you have your
target message being only partly synthesized so that one or both
primer binding sites are absent.

I usually treat my cDNA with 2U RNAse H for 20 minutes at 37 C
following cDNA synthesis.  

Hope this helps.

Good Luck!

Lauryl Nutter
Biological Sciences
University of Calgary
lmjnutte at acs.ucalgary.ca



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