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Old 09-28-2013, 12:12 PM   #1
firefly2280
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Location: London

Join Date: Oct 2012
Posts: 28
Default Struggling to amplify DNA with barcoded primers

Dear all

I am using the MiSeq for 16S amplicon sequencing with custom barcodes.

I use the barcoded primers on extracted genomic DNA. However, I have quite low concentrations of starting DNA, sometimes too low for a Qubit to detect. Some samples amplify well with the primers, some dont and some dont at all. It doesnt seem to relate to starting conc as sometimes the ones with undetectable levels amplify well.

I know that there can be random fluctuations in priming efficiency with low conc DNA samples but I dont know how to get those ones that dont amplify to work....

Ive tried touchdown PCR, enrichment PCR and varying annealing temps and template amount added but nothing works. When I do the PCR with the same primers minus the barcodes, they pretty much all work well.

Can anyone help?

Thanks
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