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Old 01-28-2010, 07:55 AM   #3
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Location: Cambridge, MA

Join Date: Mar 2009
Posts: 141

Interesting to know that the 130 bp peak is self ligated adapter. I did actually do a gel size selection step following ligation, but it seems that was insufficient to actually eliminate self-ligated adapter. I guess that migration through the gel was imperfect such that there were still some adapters "trapped" with the higher MW DNA, and that they preferentially amplified in the PCR.

Originally Posted by upenn_ngs View Post
from my experience, it is necessary to to a gel sizeselect, especially if you are doing whole exome protocol. the peak at 130 is self-ligated adapter with pcr extension. invitrogen has a great precast gel that makes the sizeselect quick. run the gel after ligation and before pcr to separate.
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