PCR based test for TruSeq library contamination in Pre-PCR lab?

lescagasan · 10-16-2013, 03:52 PM

Hi,
I am looking for a simple PCR based assay to evaluate if there is TruSeq amplified library present in our Pre-PCR lab. I would like to use the Bioanalyzer to quantitate. Would something as simple as designing primers to the P5/P7 and amplifying work? Could I just use the leftover primers from the library prep kit? How many cycles would you recommend? Any input or ideas welcomed.

Genohub · 12-14-2013, 02:04 PM

Your pre-PCR libraries probably contain doubly ligated product which in itself can amplify. How about running qPCR with some PCR amplified positive controls and comparing Ct values. I can't think of a good way for you to do with with a bioanalyzer. Are you having contamination issues?

- Genohub

ECO · 12-21-2013, 10:06 AM

I use Kapa's qPCR quantitation kit (which as you said is P5/P7 primers). Bioanalyzer won't be sensitive enough.

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10-16-2013, 03:52 PM	#1
lescagasan Junior Member Location: North Carolina Join Date: Oct 2013 Posts: 1	PCR based test for TruSeq library contamination in Pre-PCR lab? Hi, I am looking for a simple PCR based assay to evaluate if there is TruSeq amplified library present in our Pre-PCR lab. I would like to use the Bioanalyzer to quantitate. Would something as simple as designing primers to the P5/P7 and amplifying work? Could I just use the leftover primers from the library prep kit? How many cycles would you recommend? Any input or ideas welcomed.

12-14-2013, 02:04 PM	#2
Genohub Registered Vendor Location: genohub.com Join Date: Mar 2013 Posts: 210	Your pre-PCR libraries probably contain doubly ligated product which in itself can amplify. How about running qPCR with some PCR amplified positive controls and comparing Ct values. I can't think of a good way for you to do with with a bioanalyzer. Are you having contamination issues? - Genohub

12-21-2013, 10:06 AM	#3
ECO --Site Admin-- Location: SF Bay Area, CA, USA Join Date: Oct 2007 Posts: 1,358	I use Kapa's qPCR quantitation kit (which as you said is P5/P7 primers). Bioanalyzer won't be sensitive enough.