Southern Blot

Aawara Chowdhury via methods%40net.bio.net (by aawara from FEMA-trailer.org)
Mon May 28 05:05:46 EST 2007


In <5btb5iF2t3rbkU1 from mid.dfncis.de>,
 Anne <Anne.Roessger from klinik.uni-regensburg.de> wrote:

> I already tried inverse PCR like each other method (RACE, RS-PCR) and it 
> didn`t work. But I´ll try again.
> Thanks for the link to  PCRLinks.com.

Your goal is to identify the sites that your transposon is inserted into,
correct?

Well, here's a way that I've used that doesn't involve PCR, but does
require that the selectable marker on the transposon also work in
E. coli.

a) Isolate genomic DNA after transposition.
b) Cut it with a restriction endonuclease that does not cut within
   the transposon.
c) Ligate the cut genomic DNA into your favourite plasmid vector.  
   Plate the ligation on media selective for the selectable marker 
   on the transposon.
d) Isolate plasmid from individual colonies, and sequence the ends.

AC
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