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

Join Date: Mar 2009
Posts: 141
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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.


Quote:
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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