Well, if you are getting a high number of "adapter dimers" -- P1-P2 ligation products with no, or very short insert it becomes an issue. Basically they are the devil to get rid of after enrichment PCR. First they will amplify preferentially during PCR, because they are small. Second, even if you do a size selection after PCR, you can't remove all of them, because some of them will anneal back to full length library molecules.
That is why I would recommend adding a QC step after P2 ligation. If you see some P1-P2 (adapter dimer) length products at that point, I suggest an additional size selection. Ampure would probably do the trick.
In principle, you could do the size selection after enrichment PCR. But you would want to force the "hitch-hiking" adapter dimers to leave their "hiding place" by running a denaturing gel. But I have never tried this -- probably more trouble that it is worth. Multiple cycles of ampure also help. But this would be for cases where less than 50% of the amplicons are adapter dimers.
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Phillip
That is why I would recommend adding a QC step after P2 ligation. If you see some P1-P2 (adapter dimer) length products at that point, I suggest an additional size selection. Ampure would probably do the trick.
In principle, you could do the size selection after enrichment PCR. But you would want to force the "hitch-hiking" adapter dimers to leave their "hiding place" by running a denaturing gel. But I have never tried this -- probably more trouble that it is worth. Multiple cycles of ampure also help. But this would be for cases where less than 50% of the amplicons are adapter dimers.
--
Phillip
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