TruSeq with AmPure Beads in during Library Prep

Malasky53 · 08-09-2012, 05:19 AM

Hi Everyone:

We are having some issues during library prep recently and are asking for any opinions and some help out there from our fellow Illumina users. I have been doing TruSeq sample prep now for a year and have had no real issues until lately. We have started to process anywhere from 60 to 92 samples for full Exome capture. We sheer by covaris and have a starting gDNA yield of >1ug so we have enough starting material even after viewing the samples with a HS Caliper Chip. We have tried changing all of our reagents and solutions to make sure everything has been fresh and nothing expired and are still seeing a dramatic drop off after the first LM-PCR. We are using NimbleGen V3 for our capture and using NEB Phusion Master Mix for the LM-PCR. So my biggest question is that it looks like we are losing the product during the AmPure steps and was wondering if anyone leaves the beads in during the TruSeq prep and then using the Qiagen Qiaquick clean up method to get rid of the beads before capture.

Sorry about the long explanation but any help would be greatly appreciated.

niceday · 08-09-2012, 08:08 AM

I haven't been involved in next gen for almost a year but I do remember ampure beads can go off. Have you tried a new fresh aliquot of your beads? Also you could try and ph you beads and then contact your ampure rep to see if there is a problem there.

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08-09-2012, 05:19 AM	#1
Malasky53 Junior Member Location: Gaithersburg, Md Join Date: Apr 2012 Posts: 1	TruSeq with AmPure Beads in during Library Prep Hi Everyone: We are having some issues during library prep recently and are asking for any opinions and some help out there from our fellow Illumina users. I have been doing TruSeq sample prep now for a year and have had no real issues until lately. We have started to process anywhere from 60 to 92 samples for full Exome capture. We sheer by covaris and have a starting gDNA yield of >1ug so we have enough starting material even after viewing the samples with a HS Caliper Chip. We have tried changing all of our reagents and solutions to make sure everything has been fresh and nothing expired and are still seeing a dramatic drop off after the first LM-PCR. We are using NimbleGen V3 for our capture and using NEB Phusion Master Mix for the LM-PCR. So my biggest question is that it looks like we are losing the product during the AmPure steps and was wondering if anyone leaves the beads in during the TruSeq prep and then using the Qiagen Qiaquick clean up method to get rid of the beads before capture. Sorry about the long explanation but any help would be greatly appreciated.

08-09-2012, 08:08 AM	#2
niceday Member Location: cambridge Join Date: Apr 2010 Posts: 68	I haven't been involved in next gen for almost a year but I do remember ampure beads can go off. Have you tried a new fresh aliquot of your beads? Also you could try and ph you beads and then contact your ampure rep to see if there is a problem there.