pcr cloning

suter at vax.mpiz-koeln.mpg.dbp.de suter at vax.mpiz-koeln.mpg.dbp.de
Wed Sep 4 08:59:29 EST 1991



dear netters,

a lot has been said here about the cloning of PCR products,
and I have read lots of usual advice on this topic in the
last two or three years.

However, i now have a *hammer* pcr product of 1000 bp which
i have been trying to clone for 1 month, and it just won t
work !

I have tried it blunt end, with restriction enzymes, by generating
lots of colonies and then colony hybridization, etc. etc.
The vector(s) appear to be o.k.

So my questions are: do you have a suggestion what I could do ?
I must state that I have cloned "numerous" other PCR fragments
(o.k., with difficulty, but nothing like this).

Secondly: DOES ANYBODY OUT THERE KNOW WHY THIS IS SO HARD ??
What is the difference between a piece of normal DNA and a PCR
product ?
To start this discussion, here are some of my ideas:
1. dNTPs are somehow modified before incorporation (because
of extended periods at 93 degr. C ?)
2. Primers sneak into the ds DNA fragment (like when you sequence
with ds plasmid), and you get a DNA molecule with locally 3 strands
3. the fragments are not double stranded, but single stranded.
4. some other aggresive modification is taking plac ??
5. Taq Polymerase sticks to the ends of the fragment (like CIP) and is
hard to remove.
6. some component in the reaction inhibits t4 ligase

all these *brainstorm* suggestions would hamper ligation.

Perhaps you have another idea also, please post it to the net.

furious,
C. Suter-Crazzolara

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disclaimer: naturally we use only perkin elmar Taq for our reactions




(the other suppliers havent got the patent)
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