pcr cloning

Brian Hjelle bhjelle at vaxine.unm.edu
Wed Sep 4 13:39:51 EST 1991


In article <9109041300.AA06628 at genbank.bio.net> suter at vax.mpiz-koeln.mpg.dbp.de writes:
>
>
>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).

I have a radical suggestion. Put it into lambda. I don't know what
sites you have, but the ZAP 2 vector has a polylinker of about
5 sites, and probably includes a blunt end site.

Your odds should improve when you screen, say 10-30K plaques!!

>
>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 ?

I wish I did, but I don't think any of the things you suggested
are right either. I have had the same problem on several occasions.

Brian  



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