RT reagents affect PCR

Steve Hopkins shopkins at fs1.ho.man.ac.uk
Fri Dec 16 17:25:33 EST 1994


>: > 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. 

>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. 
>Loryn
> 
Thanks for the response. However, this is almost the opposite of our problem. 
We were reamplifying PCR products and found that the maximal amount of cDNA 
generated from this was much less than that from the same cDNA generated in an 
RT reaction. When we added RT regents to the PCR'd DNA it boosted the 
concentration of PCR products to the level obtained with RT cDNA as the 
starting material. When the cDNA is diluted in the PCR  buffer(with dNTP's 
etc) the drop in products reflects the dilution of the RT reagents, not the 
cDNA added. It's very odd, but reproducable. The RT itself, rather than the 
reagents, seem to contribute about half of this booster effect.  
Steve



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