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From: suttle@mbcf.stjude.org
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: PCR fragment cloning into m13 vectors
Message-ID: <1991Mar15.115833.8277@mbcf.stjude.org>
Date: 15 Mar 91 17:58:33 GMT
References: <1991Mar12.155321.1@csc.fi>
Organization: St. Jude Children's Research Hospital
Lines: 41

In article <1991Mar12.155321.1@csc.fi>, pollanen@csc.fi writes:                
>  .... If anyone has tried to introduce (PCR)
> DNA fragments into m13 vector please let me know of 
> the problems and how did you solve those problems...
> Especially talking about PCR fragment cloning into 
> m13 and also into expression vectors too.

I clone PCR fragments into M13 vectors all the time, by pretty much the
same method you are trying to use.  I design my primers so that a restriction
site is included near each end.  I can think of some problems that may be
causing your cloning difficulties:
   1.  The primers should be designed so that the restriction cleavage site
is near the center of @20mer.  If you are hoping that the enzyme will cleave 
off the very last nucleotide or two of the PCR product, you may have trouble.
       A method was described in Nucleic Acids Research 18:6156 (1990) for 
"Efficient cloning of PCR generated DNA containing terminal restriction
endonuclease recognition sites"  In this article, Jung et al. showed that
if the PCR product is generated with 5'-Phosphorylated primers and then
concatemerized with T4 DNA ligase, it is more readily digested at the
terminal restriction sites.  I have not had a reason to try this, but it
sounds reasonable.  
   2.  The M13 DNA may not be completely digested with both PstI and HindIII.
There was a BRL Focus article about double digestions of the multiple cloning
site (Focus 8:3 p. 9, 1986) in which they investigated the importance of using
the restriction enzymes in particular order.  They found that HindIII should be
used before PstI and not the reverse.  Also, it would be difficult to use them
simultaneously because their sites are so close together.
   3.  You can verify that your M13 DNA is cut by both restriction enzymes
by a simple test described in FMC Resolutions newsletter vol.2 no. 3.  It 
basically involves taking aliquots of your DNA after both the first and
second restriction digestion and digesting them together with an enzyme which
cuts outside of the polylinker - like MstI.  This produces two pieces of DNA
which differ in length by the distance between the first two sites (like
PstI and HindIII).  Separation on 4% NuSieve agarose makes it possible to
distinguish the two bands, which may be as few as 10 bp apart.  I have used
this protocol many times and would be glad to fax you a copy, if you need it.

If none of these ideas solves your problem, you could directly sequence your 
PCR product, but that involves a whole new set of problems!!!
Good luck!  -- Barbara Bugg (also M.S.)  St. Jude Children's Research
Hospital  Memphis, TN  at SUTTLE@STJUDE.ORG