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Old 01-19-2015, 08:10 AM   #1
Chief_Lazy_Bison
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Location: Ames, IA

Join Date: Dec 2014
Posts: 9
Question Nextera indexing and taq polymerases

Hello!

I am trying to sequence (Miseq) a custom amplicon from many samples using the Nextera indexes. For those of you not familiar, this involves a two step PCR protocol. The first round of PCR is carried out with your custom primers that have 5' overhangs complementary to the nextera indexes. The second round of PCR adds the indexes and adapters. The issue I am having is that my primer set contains inosine and so I can only get amplification using a Taq polymerase (Accuprime Taq in my case). This leaves my 1st PCR product with 3' A overhangs and I am not sure how this will effect the 2nd PCR step.

If I use a proofreading enzyme in the 2nd step, will this remove the 3' A's or do I need to include a specific step in my protocol to blunt my 1st round amplicons?

Could I accomplish blunting by using a proofreading polymerase and including a 72 degree incubation before the initial denaturing step in my 2nd round of PCR? Do I need to do a specific blunting step and then an additional PCR cleanup before round 2 of PCR?

Thanks!
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Old 02-05-2015, 03:58 PM   #2
ScottC
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Location: Monash University, Melbourne, Australia.

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... or, has anyone out there used regular Taq to produce libraries? I guess that would answer the question about whether it will work. I think I recall someone in a thread about 16S survey sequencing saying that they generate their amplicons using regular Taq, but I don't know what they were using for their secondary PCR step.

Cheers,

Scott.
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