Comments on: PCR – Oly RAD-seq Test-scale PCR http://onsnetwork.org/kubu4/2015/10/02/pcr-oly-rad-seq-test-scale-pcr-2/ University of Washington - Fishery Sciences - Roberts Lab Tue, 09 Apr 2019 18:37:58 +0000 hourly 1 http://wordpress.org/?v=4.0 By: Restriction Digest – Oly gDNA for RAD-seq w/AlfI | Sam's Notebook http://onsnetwork.org/kubu4/2015/10/02/pcr-oly-rad-seq-test-scale-pcr-2/#comment-2089 Fri, 09 Oct 2015 22:30:14 +0000 http://onsnetwork.org/kubu4/?p=1695#comment-2089 […] initiated the RAD-seq procedure for the sample set described below. However, the test scale PCR yielded poor results. Katherine Silliman suggested that the poor performance of the test scale PCR was likely due to low […]

]]> By: kubu4 http://onsnetwork.org/kubu4/2015/10/02/pcr-oly-rad-seq-test-scale-pcr-2/#comment-1986 Fri, 02 Oct 2015 23:41:09 +0000 http://onsnetwork.org/kubu4/?p=1695#comment-1986

Thanks for looking at this and thanks for passing along the Matz protocol! Will definitely give that a read-through.

]]> By: Katherine Silliman http://onsnetwork.org/kubu4/2015/10/02/pcr-oly-rad-seq-test-scale-pcr-2/#comment-1985 Fri, 02 Oct 2015 22:55:18 +0000 http://onsnetwork.org/kubu4/?p=1695#comment-1985 It doesn’t look good. In the Matz 2b rad protocol, they recommend keeping cycles below 15 otherwise you see a loss in heterozygosity.
https://github.com/z0on/2bRAD_GATK/blob/master/2bRAD_protocol_aug25_2014_nnrw.pdf

If the pCR isn’t working, then it’s because there aren’t many fragments with adaptors. The Matz protocol has an overnight ligation, so maybe try that?

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