Problems cutting PCR product
tvink at xs4all.nl
Tue Sep 10 02:36:24 EST 2002
Two possibilities I suppose,
1. Most likely, your restriction site is at the extreme end of your
PCR fragment and can therefor not cut. Remedy. Design primers with an
extra tail of irelevant nucleiotides or, easier in this case,
posphrylate/ligate your PCR fragment to make your site internal and
than cut , this should always work.
2. Your RE is methylation dependent. Remedy. Choose another RE.
On Tue, 10 Sep 2002 08:57:14 +0200, Trond Erik Vee Aune
<trondaun at chembio.REMOVETHISBEFOREREPLYING.ntnu.no> wrote:
>After amplyfying a 1 kb fragment I can't seem to be able to clone it
>into my vector. I tried using the same restriction enzymes on a plasmid
>and they cut with great efficiency. I could also clone the plasmid
>fragments and got thousands of colonies. So there's no problems with my
>ligation or transformation procedures.
>I know my PCR product contains the right restriction sites, so the only
>possibility left is that for some reason my enzymes will just not cut
>this template. I have heard some stories about enzymes having problems
>cutting synthetic DNA, why is that, and how can I circumvent these troubles?
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