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Old 04-23-2017, 08:19 PM   #5
Location: Honolulu, HI

Join Date: Jul 2015
Posts: 40

Originally Posted by nucacidhunter View Post
I wonder by "failure" you mean that a digested-ligated DNA PCR was successful without sized-selection and failed after size-selection. If that is the case I would column purify Pippin eluate or dilute it for PCR or use different DNA polymerase because Pippin buffer composition can inhibit some polymerases activity.
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!
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