adapter ligation - Illumina

Celia · 02-25-2011, 07:24 AM

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.

Efortier · 02-25-2011, 10:36 AM

Hi,

You could try to qPCR your libraries.

RNAseqer · 03-09-2011, 08:47 PM

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.

niceday · 03-10-2011, 02:58 AM

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.

TUCF JSS · 03-27-2011, 12:10 PM

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

greigite · 03-29-2011, 12:06 PM

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

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02-25-2011, 07:24 AM	#1
Celia Junior Member Location: Spain Join Date: Jun 2010 Posts: 3	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.

02-25-2011, 10:36 AM	#2
Efortier Junior Member Location: Montreal Join Date: Nov 2009 Posts: 7	Hi, You could try to qPCR your libraries.

03-09-2011, 08:47 PM	#3
RNAseqer Member Location: London Join Date: Sep 2010 Posts: 22	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.

03-10-2011, 02:58 AM	#4
niceday Member Location: cambridge Join Date: Apr 2010 Posts: 68	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.

03-27-2011, 12:10 PM	#5
TUCF JSS Junior Member Location: Boston, MA Join Date: Mar 2011 Posts: 8	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

03-29-2011, 12:06 PM	#6
greigite Senior Member Location: Cambridge, MA Join Date: Mar 2009 Posts: 141	Just blunt end clone your libraries & Sanger sequence a few clones each- quick way to check- cheap relative to an Illumina run!