Inverse PCR

Susanne Rohrer "srohrer(removethis)" at immv.unizh.ch
Fri Jul 21 08:09:29 EST 2000



Malay wrote:

> I am using a portion of genomic DNA eluted from the agarose gel and ligating
> it
> to from cirlcles for my template.
>
> It's a strain of Psedomonas. The fragment I am ligating is ~4 kb in length.

Theoretically it should work like this.
I've done it before without cutting out the right size band at all. I took
about 300ng of genomic DNA, digested it (overnight), ppt and ligated in a
rather large volume overnight (100ul instead of 15 for normal ligations, using
the same amt of ligase). Then I ppt again. I used Blue Dextran to get
everything down again. I resuspended in 10ul and used 1ul for a PCR reaction.
I did a touchdown.

However, at the moment I am doing it with primers designed from a very short
piece of known sequence, and I have the feeling that my primers are bad. This
could be your problem too.
I get a major band, but lots of smearing form top to bottom. I will have to
optimize it a little.

How do you clean up the fragments from the gel? Maybe you're carrying over too
much inhibiting stuff?
--
Susanne Rohrer
Institute of Medical Microbiology
University of Zurich
Switzerland

srohrer at immv dot unizh dot ch







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