Path: utzoo!utgpu!watmath!iuvax!bionet!bionet-20.bio.net!SFLANAGAN
From: SFLANAGAN@BIONET-20.BIO.NET (Steven D. Flanagan)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Cloning of PCR fragments RAY@leicester.ac.uk
Message-ID: <12485894073.20.SFLANAGAN@BIONET-20.BIO.NET>
Date: 13 Apr 89 21:44:37 GMT
Sender: daemon@NET.BIO.NET
Lines: 22


I am cloning blunt end PCR fragments (216 bp) into Bluescript
plasmid using Not1 linkers (ligated after PCR).  The yield is
very low but I have obtained 12 recombinant clones.  One problem
that I have encountered with Bluescript is that the color
selection tends to disappear with small inserts, especially if
the insert is in the reading frame.  Perkin-Elmer/Cetus Amplitaq
is highly purified and reliable. I haven't heard of exonuclease
problems with either the enzyme (J.Bacti. 127(2):1550-7) or other
commercial preparations.

If you write to me with more specific problems or questions, I
may be able to give you protocol and/or references for cloning
and screening.  Good luck!

Heidi Bauer
SFLANAGAN@BIONET-20.BIO.NET
Neurosciences Division
Beckman Research Institute of the City of Hope
Duarte, California, USA  

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