SEQanswers

Go Back   SEQanswers > Sequencing Technologies/Companies > Illumina/Solexa



Similar Threads
Thread Thread Starter Forum Replies Last Post
Library prep woes unusual bioanalyzer profile of Illumina RNA-seq library petey Sample Prep / Library Generation 1 11-19-2012 09:00 AM
Library Quantification for pooling theward Illumina/Solexa 2 10-30-2012 04:19 AM
CHIP-Seq and MBDCap-Seq Library Prep ChristmasSunflower Sample Prep / Library Generation 0 03-29-2012 01:25 PM
Pooling samples in Sanger sequencing? Alex Coventry Sanger/Dye Terminator 6 10-25-2011 03:49 AM
Pooling the samples lindenb Sample Prep / Library Generation 3 07-29-2010 11:59 AM

Reply
 
Thread Tools
Old 02-13-2013, 05:49 PM   #1
vl80
Member
 
Location: Asia

Join Date: Nov 2012
Posts: 34
Default Pooling samples for library prep using True seq

We have used barcoded primers for a PCR and our aim is to pool 8 samples together before subjecting to library prep using True seq (a modified protocol for amplicons) so that we have 8 samples per index.
Can someone tell me what amount of DNA should be pooled from each sample/
The recommendation I know is conc > 50 ng/microl and amount > 10microg per index. So, for pooling per index, does each sample need to have >10 microg per it or the 10 microg can be made up from the different samples at equal amounts (say 2 microg each).

Thanks a lot
vl80 is offline   Reply With Quote
Old 02-14-2013, 05:06 AM   #2
pmiguel
Senior Member
 
Location: Purdue University, West Lafayette, Indiana

Join Date: Aug 2008
Posts: 2,281
Default

Quote:
Originally Posted by vl80 View Post
We have used barcoded primers for a PCR and our aim is to pool 8 samples together before subjecting to library prep using True seq (a modified protocol for amplicons) so that we have 8 samples per index.
Can someone tell me what amount of DNA should be pooled from each sample/
The recommendation I know is conc > 50 ng/microl and amount > 10microg per index. So, for pooling per index, does each sample need to have >10 microg per it or the 10 microg can be made up from the different samples at equal amounts (say 2 microg each).

Thanks a lot
What "modified protocol"? How are we supposed to advise you on parameters for your protocol that we don't have?

--
Phillip
pmiguel is offline   Reply With Quote
Old 02-14-2013, 05:12 AM   #3
vl80
Member
 
Location: Asia

Join Date: Nov 2012
Posts: 34
Default

Sorry, about it. Modification is just skipping the fragmentation but following the other steps of Truseq.
vl80 is offline   Reply With Quote
Old 02-14-2013, 07:24 AM   #4
pmiguel
Senior Member
 
Location: Purdue University, West Lafayette, Indiana

Join Date: Aug 2008
Posts: 2,281
Default

So you are ligating Y-adapters onto PCR products? I guess as long as your are coming into the ligation with at least 100 ng of DNA total (that is nanograms, not micrograms) you will probably be fine.

Not sure where you get the >10 ug/index figure. TruSeq DNA libs want 1 ug, not 10.

--
Phillip
pmiguel is offline   Reply With Quote
Old 02-14-2013, 12:03 PM   #5
vl80
Member
 
Location: Asia

Join Date: Nov 2012
Posts: 34
Default

Thank you very much. Yes, I read that in Trueseq the requirement is for 1ug. The tests would be done for us for the first time at a commercial venture. Thier requirement states that the conc of DNA should be > 50ng/ul and the total amount to be > 10ug :/
vl80 is offline   Reply With Quote
Old 02-14-2013, 02:16 PM   #6
kcchan
Senior Member
 
Location: USA

Join Date: Jul 2012
Posts: 182
Default

This is something you should talk to your service provider about. The 10ug spec is the Illumina standard for their official service providers; it's only intended for genomic DNA. Since your samples are not gDNA, you will need to speak with your provider to find out how much you will need to submit. In your case, you probably don't need to provide very much sample as your libraries will only require adapter ligation. You should also ask them about using a PCR free protocol to eliminate any bias in your pool.
kcchan is offline   Reply With Quote
Old 02-14-2013, 02:25 PM   #7
vl80
Member
 
Location: Asia

Join Date: Nov 2012
Posts: 34
Default

Thank you very much for both suggestions. Would do so.
vl80 is offline   Reply With Quote
Reply

Tags
pooling samples

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 04:41 PM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2018, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO