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Old 08-11-2017, 12:42 AM   #1
Location: Poland

Join Date: May 2016
Posts: 10
Default Illumina 16s protocol: size


I've been having a hard time amplifying 16s DNA from active sludge with Illumina suggested primers v3-4.

I tried NebNext HIFI and iProof mixes, same results.
Anyone knows why?

I am using standard conditions, but I never get the 550 bp band after 1st PCR, nor the 630 bp band after 2nd. I tried qPCR, and after 25 cycles suggested by Illumina it was empty (started rising at about 27 cycels), so I switche do 32 cycles of 1st PCR. Getting a lot of different products but not the 550 band.

Do the active sludge bacteria have a different size of this region? Then maybe I am throwing it away with 0,8x AMPure X beads after 1st PCR (it is not well visible on the gel before purification either). Or do the primers simply not hybridise?

I would be grateful for a quick answer.
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