Path: utzoo!utgpu!watserv1!watmath!uunet!bionet!bio.embnet.se!sunic!news.funet.fi!hydra!cc.helsinki.fi!nphi!msalminen
From: msalminen@nphi.fi
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: PCR fragment cloning into m13 vectors
Message-ID: <387.27e104a7@nphi.fi>
Date: 15 Mar 91 17:30:15 GMT
References: <1991Mar12.155321.1@csc.fi>
Organization: National Public Health Institute, Finland
Lines: 38

In article <1991Mar12.155321.1@csc.fi>, pollanen@csc.fi writes:
> Hello
> 
> Can anyone help me with m13 cloning I have some problems
> with ligation a PCR product into m13 vector (m13mp18)
> The size of fragments is varying from about 100bp to
> 300bp size in lenght. I have opened the m13 vector 
> by pstI /HindIII double digestion and also I have 
> digested these PCR products same way because we have
> planned primers so that PCR is (probably) producing
> pstI and HindIII sites to the ends or near of the ends
> of these fragments.... If anyone has tried to introduce 
> 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...
> 
> Sincerely yours Raimo Pollanen (M.S.)
> 
> Pollanen@CSC.FI

Hi Raimo!

You could try two things:

a) after PCR add 5-10 u Klenow. This repairs the ragged ends sometimes left by
Taq. Then precipitate and digest. Precipitate again and clone. If you do a
Kinase reaction the yield of inserts is better. Check the size of pcr-product
before cloning by gel-electrophoresis.

b) Do the fill-in with Klenow and precipitate. Blunt-end clone in suitable site
such as Sal1. Again, kinase gives better yield. 


If you need more details, feel free to contact me at msalminen@finnphi (bitnet)
or msalminen@nphi.fi (Internet) or nphi02::msalminen (Decnet)

Mika Salminen KTL, HIV-Unit.