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Old 12-01-2011, 10:35 AM   #1
jlove
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Default Using TruSeq adapters with older protocols- not working?

We have tried a couple of times to use old stand-by homemade library protocols (which worked fine with the old adapter oligo) with TruSeq adapters swapped in for the old adapters. It never seems to work right. I can't really figure out what would be different about the new adapters in terms of ligation - the libraries are end-polished and A-tailed just the same as before. So I called Illumina to ask them if there were any differences, and they would only say that it was "proprietary," and that the new ligation conditions in the TruSeq kit were optimized for the new adapters. They were not too surprised that it wasn't working using the old kit, but I can't for the life of me figure out a reason why. Anybody have any thoughts?

The old ligation protocol was 15 min at room temp using a Quick Ligase from Promega. The TruSeq kit calls for a 10 minute incubation at 30C using a secret "ligation mix."
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Old 12-01-2011, 11:44 AM   #2
Jon_Keats
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Are the adaptors purchased from Illumina or self synthesized? We order the TruSeq adaptors from IDT for CHIP-seq libraries and they seem to work very well using the NEB library kits uses a 15 min incubation at 20degrees
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Old 12-01-2011, 12:11 PM   #3
jlove
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We have both the kit version and the homemade version. They are equivalent, but I believe the testing was done with TruSeq adapters. Curious! Thanks for the reply.
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