Hi everyone!
The enzyme in my SuperScript™ II Reverse Transcriptase (Invitrogen) kit has run out and I don't have time to order a new one as I have to finish the experiments asap. Will I run into any problems if I use the same ingredients in the Superscript kit and use the "MultiScribe reverse transcriptase" from the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) instead of just the enzyme? I will do RT-PCR from cDNAs.
In fact, I ran 3 samples in qPCR by just changing the enzyme and applying it to everything else in the same way, and there was no difference compared to the other samples. However, Multiscribe works at 37C and Superscript at 42C. If I use Multiscribe, would it be better to set the temperature to 37C as well? Before I run other samples, I want to make sure that this will not affect my experimental results.
Thank you so much,
Elif
Dear @Elif Sanli,
if you are just doing some try outs or pre-experiments this might work. But if you get into real experiments with reproduction make sure you use as indicated by the manufacturer. So no changing of components or enzymes. These have been manufactured to work in certain limits of reproducibility.
Best Boris
Well, you have to follow the manf. instructions first, then if it did not work, u have to modify the ingredients and the TM.
Hi,
I would agree with Boris.
Not only are the enzymes designed to work within their own kit, there is no way of predicting how changing buffers etc. could affect performance.
I have previously compared several RT kits alongside each other on the same samples and even using all the 'proper' kit reagents I saw quite large differences in efficiency of conversion across different genes (from no difference for some genes, up to 2-3 cycles in some cases). So even if it appears to be giving similar results for one gene, you can't guarantee that will be the case for all genes.
I'd stick to one enzyme if you can, particularly if you plan to publish!
Hope that helps,
Ben.
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