PCR fragment cloning into m13 vectors

msalminen at nphi.fi msalminen at nphi.fi
Fri Mar 15 12:30:15 EST 1991


In article <1991Mar12.155321.1 at csc.fi>, pollanen at 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 at 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 at finnphi (bitnet)
or msalminen at nphi.fi (Internet) or nphi02::msalminen (Decnet)

Mika Salminen KTL, HIV-Unit.



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