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."
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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