DNA crosslinking on nylon memberane

[Kyle Legate]

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.