Southern Blot

peter via methods%40net.bio.net (by peter.ianakiev from gmail.com)
Thu May 24 11:09:14 EST 2007


On May 24, 11:55 am, "Anne" <Anne.Roess... from klinik.uni-regensburg.de>
wrote:
> Thank you Peter for your help.
> I´ll try to find the sequences next to my sensor then by RACE or
> restriction-site PCR. I try to find the integration site of my used
> transposon. If anybody does know another method to find the integration
> sites it would be very helpful for me.
>
> "peter" <peter.ianak... from gmail.com> schrieb im Newsbeitragnews:1180017336.890275.280240 from o5g2000hsb.googlegroups.com...
>
> > On May 24, 2:50 am, "Anne" <Anne.Roess... from klinik.uni-regensburg.de>
> > wrote:
> >> By PCR? You cut the nylonmembrane and run a PCR with it or how do you do
> >> it?
>
> >> "peter" <peter.ianak... from gmail.com> schrieb im
> >> Newsbeitragnews:1179930831.833752.233410 from g4g2000hsf.googlegroups.com...
>
> >> > On May 23, 8:29 am, "Anne" <Anne.Roess... from klinik.uni-regensburg.de>
> >> > wrote:
> >> >> Hello,
> >> >> does anybody know if it is possible to get the DNA out of the
> >> >> nylonmembrane
> >> >> after crosslinking ? I have to know the sequences next to the binding
> >> >> site
> >> >> of my sensor.
>
> >> >> Thanks a lot
> >> >> Anne
>
> >> > Yep, by PCR....
>
> > That's right Anne - cut the membrane with your fragment on a small
> > pieces and then use it in a PCR with your favorite primers. Just be
> > careful to wash out all the SDS which will inhibit PCR reaction even
> > at very low concentrations. Also for "crosslinking" might be better if
> > you just use NaOH rather than UV, but that will not interfere with the
> > PCR if your amplicon is small.
> > Good Luck!
> > PS. Sorry to the group for the multiple posts earlier - it is now
> > obvious that I am using Windows PC....

Just ligate and adaptor to your DNA, before you load on the Southern,
use NaOH/SSC for transfer, (No UV). PCR the fragment using the  method
above with the adaptor primer, make a library that you can screen with
a portion of your transposon. Sequence positive clones - see
integration site.
Peter



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