SEQanswers

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



Similar Threads
Thread Thread Starter Forum Replies Last Post
library prep problem ajthomas 454 Pyrosequencing 21 02-23-2012 03:12 PM
Library PCR enrichment questions (with image!) ZAAB Sample Prep / Library Generation 6 06-07-2011 06:47 PM
Rapid library prep and Nimblegen enrichment Tom Haltern 454 Pyrosequencing 1 08-05-2010 05:55 AM
Enrichment with AGILENT's Sureselect target enrichment system dottomarco 454 Pyrosequencing 1 11-18-2009 01:14 AM
Poor enrichment...defective enrichment beads? njae4 454 Pyrosequencing 0 03-09-2009 06:23 AM

Reply
 
Thread Tools
Old 10-26-2011, 12:46 PM   #1
meganathan.pr
Junior Member
 
Location: Starkville

Join Date: Oct 2011
Posts: 3
Thumbs up Problem in library enrichment

Hi,
I got a problem in my library preparations.
I tried to prepare 180/230 bp libraries. I have used NEB reagents and illumina PE adapters and primers for my library preparations.
I am getting the expected size fragments after PCR enrichment. However, the concentration is too low, ~10 ng/ul. I have repeated the same many times but did not get a high yield.
Could anyone please tell me what should I change to get high yield?

Thanks.
__________________
Meganathan
meganathan.pr is offline   Reply With Quote
Old 10-26-2011, 12:53 PM   #2
NextGenSeq
Senior Member
 
Location: USA

Join Date: Apr 2009
Posts: 482
Default

10 ng/ul is enough to sequence. When you dilute a library to 10 nM it is typically about 2 ng/ul
NextGenSeq is offline   Reply With Quote
Old 10-27-2011, 05:04 AM   #3
pmiguel
Senior Member
 
Location: Purdue University, West Lafayette, Indiana

Join Date: Aug 2008
Posts: 2,317
Default

And the new, v3, cluster kits offer an option of using 2 nM libraries. That should be available on HiSeqs and HiScanSQs. (Not sure about GAs).

--
Phillip
pmiguel is offline   Reply With Quote
Old 10-27-2011, 11:10 AM   #4
meganathan.pr
Junior Member
 
Location: Starkville

Join Date: Oct 2011
Posts: 3
Default

Thanks for your reply.
As a beginner, I would like to know your experiences. Is it not looking odd to get such a low amount of amplification when I am using 5 ug of gDNA for my library preparations? or is it normal to get such a low amplification?
__________________
Meganathan
meganathan.pr is offline   Reply With Quote
Old 10-28-2011, 03:26 AM   #5
pmiguel
Senior Member
 
Location: Purdue University, West Lafayette, Indiana

Join Date: Aug 2008
Posts: 2,317
Default

Yes, but there are lots of things that can go wrong.

First, my experience is that most gDNA concentrations estimation are done by UV spec, which tends to mean that they are a 10-100x overestimate of the true amount of DNA. This is because most genomic DNA preps are largely RNA (possibly degraded with RNAse--but that has little effect on the amount of UV the RNA absorbs.)

So there is that. Past that, the biggest issue is that contaminants in the DNA prep (ethanol, etc.) can inhibit end-repair post-shearing. That can drastically lower yields.

--
Phillip
pmiguel is offline   Reply With Quote
Old 11-07-2011, 06:01 PM   #6
sehrrot
Member
 
Location: USA

Join Date: Jul 2010
Posts: 58
Default

though you got lower than 2nM, you would change conditions for template preparation, which should end up at 12 pM-ish. So, it would not be a problem, but you may concern that your library would have lower complexity.
sehrrot is offline   Reply With Quote
Old 11-09-2011, 01:07 AM   #7
Liting
Member
 
Location: China

Join Date: Jul 2011
Posts: 12
Default

Quote:
Originally Posted by sehrrot View Post
though you got lower than 2nM, you would change conditions for template preparation, which should end up at 12 pM-ish. So, it would not be a problem, but you may concern that your library would have lower complexity.
Hello,sehrrot.I also have a problem about the PCR richment during library preparation。I also have got some unexpected fragments which are about 100bp smaller than the expected fragments after PCR。 Have you got the same problem?What should I do to avoid the problem?Thank you very much!
Liting is offline   Reply With Quote
Reply

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 08:39 PM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2020, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO