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Old 07-13-2016, 10:49 PM   #10
Location: college station

Join Date: Jan 2012
Posts: 22

Originally Posted by nucacidhunter View Post
DNA migration speed and hence calculated size is affected by presence of salts and protein in the sample. For fair comparison fragmented input DNA and PCR product has to be resuspended or eluted in the same buffer.

If you clean the PCR product with lower ratio of bead to amplicon (for instance 0.65x) short library fragments below 300bp will be cut off and that will shift the remaining library fragments peak to higher value. But I do not think that is what you want and is not advisable as it will reduce library diversity and yield.
Thanks! I will try cleaning with 1X beads then to see if the size becomes expected.

Another question though is the double peaks after ligation. From previous posts some seems to suggests that the lower peak around 300bp represent fragments with a single adapter ligated, while the very "long" fragments are caused by Y-shaped adapters on both sides. Does this suggest insufficient adapter input or poor ligation in my case since the lower peak is quite high?
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