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Old 01-29-2014, 06:25 AM   #1
flobpf
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Location: USA

Join Date: Apr 2010
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Post Designing P1 P2 adapters for RAD-seq

Hi,

I am doing some research on how to design the optimal P1 and P2 adapters for RAD-seq, and had a couple of questions.

I know the P1 adapter consists of 5'-ForwardAmplificationPrimerSite (FAPS) -- IlluminaSequencingPrimerSite (ISPS) -- Barcode-3'. However, I am not sure about the following:

1) How long do the barcodes have to be? Is there a software for designing these adapters for RAD-seq? What is the total length of the P1 adapter?

2) I assume that other than the barcode, everything else in the P1 adapter would be constant across all experiments. What is the sequence of the FAPS and the ISPS, or where can I find it?

3) Does the P1 adapter end with the overhang for the specific restriction enzyme I would be using?

4) Is there a commercial vendor for such adapters, or do we have to synthesize them base by base?

5) What is the sequence of the P2 adapter? Is it always the same across all experiments or does it have to be modified every time?

Thanks

EDIT: Also, is there a way to check at the end of the library prep that things have gone smoothly? Some PCR based method perhaps with the P1 and P2 adapters?

Last edited by flobpf; 01-29-2014 at 08:06 AM. Reason: additional question
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