c.t.dolphin at qmw.ac.uk
Wed Feb 1 11:53:05 EST 1995
Subject: Clontech libraries
From: SBSHEPARD, sbshepard at aol.com
Date: 31 Jan 1995 04:17:05 -0500
In article <3gkv6h$sen at newsbf02.news.aol.com> SBSHEPARD, sbshepard at aol.com
>Recently, I have spent a lot of time screening for numerous genes in a
>Clontech cDNA library. The inserts are supposed to be excised with
> Unfortunately, 50% of the clones that I have purified do not seem to be
>excised, despite cutting of the vector (I can see two bands after
>digestion). I am concerned about the quality of the library, given these
>problems with the linker sequences. I do not want to cut with Hind III
>and Ava 1. Has anyone else had similar problems?
Yes, the EcoRI sites were missing from a Clontech human fetal liver cDNA
gt11 library that I used a few years ago - it looked as though some exo'
activity may have resulted in blunt end ligation.
PCR the insert with vector-specific primers and subclone - use Pfu pol or
similar and do at least two independent PCRs if you're worried about
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