Inverse PCR

Susanne Rohrer "srohrer(removethis)" at
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

srohrer at immv dot unizh dot ch

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