DNA crosslinking on nylon memberane

dprieto2006 from gmail.com via methods%40net.bio.net (by dprieto2006 from gmail.com)
Thu Jul 24 13:45:28 EST 2008


On 18 jul, 15:45, Kyle Legate <lega... from hotmail.com> wrote:
> Tracy Chow wrote:
> > Hi Rory,
>
> > Thanks for your suggestion...really appreciate.
>
> > May I ask if you use Hybond-N+ or Hybond-XL (also positively charged)
> > membrane?  May I ask for the conditions you dry the membrane (room
> > temperature  or vacuum)?  How long do you *dry the membranes *prior to
> > hybridization?  How long do you *pre-hybridize* prior to probe incubation?
>
> > THANKS!!!
>
> > Tracy
>
> I'm not Rory, but I'll toss in my 0.5 cents anyway. Both membranes will
> be fine, but you crosslink them differently. N+ is nitrocellulose, XL is
> nylon, so heat or UV, respectively. Dry the membrane on some filter
> paper or other uncontaminated holding device by RT evaporation until
> there's no visible moistness before proceeding. The time it takes to
> clean up the blotting set-up should be enough time. If protected in a
> plastic sleeve or a book they can be stored dry for months before
> hybridizing, or they can be used immediately after crosslinking (~10
> minutes after obtaining them). I used Church buffer for a pre-hyb
> solution and 1 hour in a hybridization oven (65C) was sufficient. Enough
> time to label your probe.

Hi, you can also fix your nucleic acids by means o MW radiation using
a common microwave oven.
See this paper: Nucleic Acids Res. 23:879-880, 1995
hope this can help,
Daniel (dprieto from fcien.edu.uy)


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