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Old 02-25-2011, 07:24 AM   #1
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Location: Spain

Join Date: Jun 2010
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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.
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Old 02-25-2011, 10:36 AM   #2
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You could try to qPCR your libraries.
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Old 03-09-2011, 08:47 PM   #3
Location: London

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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.
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Old 03-10-2011, 02:58 AM   #4
Location: cambridge

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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.
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Old 03-27-2011, 12:10 PM   #5
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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
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Old 03-29-2011, 12:06 PM   #6
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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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illumina, ligation

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