Go Back   SEQanswers > Applications Forums > Sample Prep / Library Generation

Similar Threads
Thread Thread Starter Forum Replies Last Post
Rapid Library Library Quantitation using concentration not RFU GraemeFox 454 Pyrosequencing 9 10-21-2011 12:27 AM
Help with denaturing low concentration Illumina PE Library TonyBrooks Sample Prep / Library Generation 3 09-27-2010 09:30 AM
Low concentration GA library Melanie Sample Prep / Library Generation 4 08-04-2010 02:34 PM
low cDNA concentration after the RAPID protocol dina 454 Pyrosequencing 0 07-11-2010 12:48 PM
Anyone use low concentration for ChIP-seq prep? Audry888 Sample Prep / Library Generation 4 05-28-2010 12:37 PM

Thread Tools
Old 08-11-2010, 07:07 AM   #1
Senior Member
Location: Boston area

Join Date: Nov 2007
Posts: 747
Default Better to risk low concentration library or further amplification?

Here's an issue which has arisen with some SureSelect processed libraries for the Illumina: after the hybridization the amount of library is barely within spec -- hence my question about quantitation.

So, the question is which path to take: push the libraries forward as is, with a risk of missing peak sequencer yield due to suboptimal cluster density, or run a few more (how many?) cycles of PCR to increase the concentration but also up the odds of getting PCR duplicates?
krobison is offline   Reply With Quote
Old 08-11-2010, 07:10 AM   #2
Senior Member
Location: USA

Join Date: Apr 2009
Posts: 482

It worked fine for me and I could barely detect it on my Nanodrop. We are in the process of evaluating the Kapa Q-PCR kit which I think the Broad Institute has switched to.
NextGenSeq is offline   Reply With Quote
Old 08-12-2010, 08:09 AM   #3
Senior Member
Location: Oklahoma

Join Date: Sep 2009
Posts: 411

Check out this thread.
You can prep a flowcell with a library that is <100pM with no problems.
GW_OK is offline   Reply With Quote
Old 08-12-2010, 02:40 PM   #4
Senior Member
Location: Boston

Join Date: Feb 2008
Posts: 693

No, I think the expected PCR duplicate rate is irrelevant to the PCR cycles. The duplicate rate is determined by the number of molecules before amplification.
lh3 is offline   Reply With Quote

Thread Tools

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off

All times are GMT -8. The time now is 06:10 PM.

Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2022, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO