RT reagents affect PCR

Loryn Sellner lsellner at uniwa.uwa.edu.au
Tue Dec 13 21:24:12 EST 1994


Michael R. Frank (mr-frank at uiuc.edu) wrote:
: shopkins at fs1.ho.man.ac.uk (Steve Hopkins) wrote:
: >
: > While developing a quantitative PCR method we have become aware that transfer
: > of small amounts of the cocktail of reagents in the reverse transcriptase
: > reaction (many of which which are present at much higher concentration than
: > those in the PCR-mix) influences the subsequent PCR. This means that the PCR
: > signal may be a function of the amount of RT-mix transferred, as much as the
: > amount of cDNA. It could also be particularly important where cDNA standards, 
: > not in RT-mix cocktail, are used. Has anyone experience of what RT-mix factors 
: > might be important? Can transfer of random primers affect PCR? We are checking 
: > some of these things out, but any help would be appreciated


Steve,
I spent quite a lot of time setting up a one tube RT-PCR, and found that 
if you have too much of the RT itself in the mix, it inhibited Taq. The 
ratio of taq:RT you needed to keep below was about 2U:3U ( I used 2U Taq 
to 0.5 U RT). If you heat inactivate the RT between the cDNA synthesis 
and adding the Taq in the PCR then it didn't affect it. Also having 
random primers present interfered with the PCR, I used the same primers 
to initiate CDNA synthesis as for PCR.
Hope this helps,
Loryn
 



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