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From: fish@LINDA.LLNL.GOV (chris)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: PCR fragment cloning into M13 vectors
Message-ID: <9103122139.AA01190@linda.bio386>
Date: 12 Mar 91 21:39:56 GMT
Sender: daemon@genbank.bio.net
Lines: 23

RP>Can anyone help me with m13 cloning I have some problems
RP>with ligation a PCR product into m13 vector (m13mp18)
RP>The size of fragments is varying from about 100bp to
RP>300bp size in lenght. I have opened the m13 vector
RP>by pstI /HindIII double digestion and also I have
RP?di>tested these PCR products same way because we have
RP>planned primers so that PCR is (probably) producing
RP>pstI and HindIII sites to the ends or near of the ends
RP>of these fragments.... If anyone has tried to introduce
RP>DNA fragments into m13 vector please let me know of
RP>the problems and how did you solve those problems...
RP>Especially talking about PCR fragment cloning into
RP>m13 and also into expression vectors too...

An alternative and efficient cloning method for PCR products was developed by Aslanidis
and de Jong (see Nucleic Acids Research, 18:6069-6074).  The method does not use
ligation, and the cloning efficiency is extremely high (only recombinants are
produced).  As mentioned by John Nash in an earlier response, small inserts give
light blue colonies when a pUC-based vector is employed. 

Chris T. Amemiya
Lawrence Livermore National Laboratory
fish@amoeba.llnl.gov