My samples seem to have similar levels of amplification with no primer clouds or primer-dimers and no secondary amplification. Is there a reason I need to magnetic bead clean up each sample right after PCR instead of doing them all together after quantification/dilution/pooling?
Thanks!
Somehow you have your answer in question itself. We clean first PCR product seperately because they are not barcoded. And we clean second PCR seperately because for pooling all barcoded samples must be normalized. I dont see any other way to do this. And there isnt any logic to do it the other way round. Can you suggest your idea what and why you think for doing cleanup after pooling?
Our protocol uses only 1 PCR reaction with barcoded primers.
Qubit should be able to accurately quantify PCR product without clean up, regardless of dNTPs floating around. Thus, shouldn't we be able to dilute them each to the same concentration, pool and then do one clean up?
what protocol do you use? could you please share it here or via message?
Hey David, I think the main reason you need to do PCR--> purification --> qubit --> pooling is that you want to get an accurate reading on your final output (purified PCR), from there you can ensure you pool equal morality of each indexed samples. Of cause this is assuming you want same amount of reads for each of you samples.
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