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Old 05-01-2017, 10:57 AM   #7
ymilesz
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Location: Orlando

Join Date: May 2017
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Quote:
Originally Posted by Carcharodon View Post
Well, I originally followed the protocol laid out by Peterson et al. (2012), where digested DNA with ligated adapters were size-selected (w/ Pippin-Prep) and then went through PCR.

3 or 4 out of my 6 total libraries showed amplification. The other two did not. So the eluate itself didn't seem to be inhibiting PCR. I suspect that there was just very little DNA in the size-selected DNA for those 2 - 3 libraries. Which is one reason I was considering running a PCR before size-selection.

I'm taking steps to ensure that won't be the case here, but you never know. At least now I have the benefit of experience!
Hi I am also following the ddRAD protocol from Peterson et al 2012, and I too am having trouble with PCR after size selection. Are you doing the 20uL reactions as suggested as well?
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