Fixing oligonucleotides on N+ membranes

Dr. Hedley Carr bmbthc at removebmb.leeds.ac.uk
Fri Jan 21 11:17:27 EST 2000


Dear January,
Oligos should blot (and stick) onto Nylon just fine - you might want to
try 
more positively charged membranes such as Amersham's 'Hybond N+' or 
Schleicher & Schuell's 'Nytran SuperTrans' (I think the name is right!).
Anyway, I've been doing sequencing using both these membranes as support
and
my DNA sticks just fine. Perhaps you're overdoing the UV treatment (i.e:
damaging the oligos excessively so that they can no longer be detected?)

I blot onto the membrane (20 min, no longer otherwise small fragments
tend
to go right through). 
Then dry in a 37 incubator with fan for 15min (DNA side up).
Then UV crosslink in a standard crosslinker (254nm) on the DNA side only
(The default setting on our machine seems to work really well, which is:
120,000 microJoules per square cm., which takes around 20 seconds of
exposure).

Et voila, your oligos should be immobilised and detectable.

Hope this helps.

Hedley Carr.
University of Leeds.
_____________________________________________

January Weiner wrote:
> 
> Hello,
>         I'm looking for a method of fixing oligonucleotides on nylon (or,
>         actually, any kind of) membranes. UV-crosslinking seems to be
>         unsufficient, as the membrane appears empty after hybridization.
>         It would be a great help for me if you could post any links or
>         hints you know about on this subject!
> 
>                 Regards,
>                 January
> 
> --
> ----)-\//-///-----------------------------------January-Weiner-3-------
> I have wrestled with reality for twenty-five years and I'm happy to state that
> I finally won over it.




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