Hey guys,
I ran a SuperScript III first strand synthesis for RT-PCR with random hexamers, followed by GoTaq DNA Polymerase PCR with dNTPs + primers to amplify my gene of interest. However, I was wondering if I should have done a PCR purification kit prior of going into GoTaq PCR.
Hi,
no you do not need to purify your cDNA prior to PCR. Just make sure, that the input volume of cDNA reaction does not exceed the maximum recommended for the kit
Best Sven
If it is possible tht there is DNA in your sample after RNA isolation, you should perform this purification step. However, most column-based RNA isolation kits include a DNase step or methods to remove DNA from your sample. Moreover, the rest of random hexamers in this sample should not lead to influencing effects on further analyses.
Thus, an aditional purification step should not be absolutely necessary.
Hi William,
You do not need to purify your ssDNA post first strand synthesis as all these kits come with compatible buffers and salt concentration which will not affect your downstream analyses. However, as Andreas Eisenreich correctly said, you need to keep a check on the gDNA contamination as that will skew your results. This can be merely monitored using a No template control (NTC set similar as your RT reaction except the reverse transcriptase) in qPCR. And in case if you find an amplification with NTC you should use the purification step or you can re-perform the DNAse and column purification of your input RNA and then perform first strand synthesis.
Hi all,
as ong as the primers are mRNA specific and do not bind/ detect gDNA this would not be needed at all. In general if this is the case it would be easier to remove gDNA upfront or to use an RT kit with included DNAse digest
Best Sven
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