SEQanswers

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



Similar Threads
Thread Thread Starter Forum Replies Last Post
Poor Ligation Efficiencies on illumina Libraries mollusc Illumina/Solexa 16 10-05-2011 06:04 AM
Adapter Ligation Question johnmillsbro Illumina/Solexa 3 07-13-2010 09:09 AM
Ratio in Adapter-Ligation mestro2 Sample Prep / Library Generation 0 05-27-2010 04:29 AM
help: time of adapter ligation lvxiaobao Sample Prep / Library Generation 1 12-24-2009 07:37 AM
solexa adapter ligation question seqgirl123 Sample Prep / Library Generation 2 10-13-2009 12:08 PM

Reply
 
Thread Tools
Old 02-25-2011, 07:24 AM   #1
Celia
Junior Member
 
Location: Spain

Join Date: Jun 2010
Posts: 3
Default adapter ligation - Illumina

How can I know if the adapter ligated?

For three of my samples (we are 6-plexing) I forgot to anneal the adapters, however all the samples turned out really well and would be ready for a lane in Illumina. The other three samples are ligated with adapter we had a for a long time and have been working perfectly.

Is there any way to find out if the adapters are ligated anyways before running a lane on Illumina?
Thank you very much.
Celia is offline   Reply With Quote
Old 02-25-2011, 10:36 AM   #2
Efortier
Junior Member
 
Location: Montreal

Join Date: Nov 2009
Posts: 7
Default

Hi,

You could try to qPCR your libraries.
Efortier is offline   Reply With Quote
Old 03-09-2011, 08:47 PM   #3
RNAseqer
Member
 
Location: London

Join Date: Sep 2010
Posts: 22
Default

i wouldn't do the run with non-annealed adapters. it simply won't work. If you're not sure whether you annealed them or not do a qPCR with good positive and negative controls. you'll get your answer right away.
RNAseqer is offline   Reply With Quote
Old 03-10-2011, 02:58 AM   #4
niceday
Member
 
Location: cambridge

Join Date: Apr 2010
Posts: 68
Default

you can also look at the bioanalyser plots. The libraries will be bigger after primer ligation. But as previously said qPCR will give you a definite answer.
niceday is offline   Reply With Quote
Old 03-27-2011, 12:10 PM   #5
TUCF JSS
Junior Member
 
Location: Boston, MA

Join Date: Mar 2011
Posts: 8
Default

Even a simple PCR and gel will give you an answer if you do not want to go through the time and expense of a qPCR or bioanalysis for just a few samples. Your sample size should be ~120bp larger than that of its previous size selection, and only amplifiable DNA (successfully ligated) will be present. If your gel is blank then ligation was unsuccessful. But it is not worth risking a lane's worth of sequencing without SOME kind of validation
TUCF JSS is offline   Reply With Quote
Old 03-29-2011, 12:06 PM   #6
greigite
Senior Member
 
Location: Cambridge, MA

Join Date: Mar 2009
Posts: 141
Default

Just blunt end clone your libraries & Sanger sequence a few clones each- quick way to check- cheap relative to an Illumina run!
greigite is offline   Reply With Quote
Reply

Tags
illumina, ligation

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 01:02 AM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2017, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO