problem in PCR clonong

Your Name email at domain.com
Tue Aug 5 00:44:40 EST 1997


In article <5rqf9v$c88 at sifon.cc.mcgill.ca>, czlq at musica.mcgill.ca wrote:

> I have been stoped by a simple PCR cloning work.I have chosen pCR.script
and pBluescript vectors and cloned my PCR fragments 
> into the vector. Everytime I got some white colones ,but none of them
have insert.  I was wondering why the  ratio of this false 
> conoles  appeared so high ? The PCR product I used are either the
original PCR product or PCR product    purified  by ethanol 
> precipitation .If anybody can help me solving this problem, I will very
appreciate. Thanks a lot in advance.
> 
> 
> lee 

lee,
I hope you are aware of the fact that the oligos generally have no 5'
phosphate, such that if you dephosphorylate your vector efficiently,
without phosphorylating your PCR product or primers, it isnt possible for
you to get any recombinants. This may seem like something everyone knows,
-but when I first arrived in my lab, the first thing I did was to try to
clone a PCR product, noone in my lab knew of the lack of 5'Phosphates, and
as a result I wasted a considerable amount of time trying to do something
everyone in the lab knew was really easy. (somewhat disheartening) Once I
knew this, I chucked in an enormous molar excess of the PCR product (with
no 5' Phosphates) with a SmaI linearised Pks (not dephosphorylated), SmaI,
and T4 DNA Ligase all in the same tube. Left overnight, replenished the
enzymes and ATP a couple of times, and then just transformed the whole
lot. -Got 1 in 3 recombinant, not a great frequency, but on my first
attempt, and using no more than one tube and minimal effort. -One note
though, have since learned that SmaI is not a very good enzyme to do this
with, apparently it can chew off a couple of nucleotides at the end, so if
the frame of your PCR product is important, you might be better off with
EcoRV. 
This is essentially the same strategy as in Stratagene's PCR cloning kit,
except that utilises an 8bp blunt cutting restriction enzyme (Srf1, I
think, same sequence as SmaI, but with an extra c and g, I think,) and Pks
engineered to contain this site in the polylinker. I kind of got the idea
of my own cloning strategy from the Stratagene adverts. Enzymes I used
were from biolabs, -used buffer 4 (reccomended for SmaI) supplemented with
something like 1 mM ATP, though supposedly for blunt end ligation its
better to lower the ATP concentration.

whatever,
hope your cloning works,

Martin

(mjakt at hkusua.hku.hk)



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