Northern hybridisation

Dr Patrick HJ Falckh p.falckh at rmit.edu.au
Wed Dec 13 21:37:32 EST 1995


I've found Hybond N+ (Amersham) to work well and have had little 
problems with background.  Provided the sample is crosslinked by UV (a 
couple of minutes on a transluminator works OK) you can strip the 
menbrane for reprobing.

	The method I use was devised back some time ago as some published 
methods weren't working for me. The prehyb and hyb solns are the same.

																													Vol							Final Conc.
Deionised formamide								10.0 ml							40 %
100 X Denhardt's												0.8 ml							3.2 X
20 X SSC																				5.0 ml							4.0 X
0.5M NaPO4 (pH 6.5)									2.0 ml							40 mM
50% Dextran Sulfate									5.0 ml							10 %
Herring Sperm DNA	(5mg/ml)		1.25 ml						250 ug/ml
SDW																									0.95 ml				
																											--------
																											25.0 ml

Prehyb for ~6 hrs @ 42C in 0.1ml/sq.cm membrane of above soln (can use 
less if using a hyb chamber and rotating containers).  After the prehyb 
pour a small amount out into a sterile 50 ml polyprop tube, add 
radiolabelled probe (~1 mill CPM/ml of hyb soln) then return to 
container/bag for hybridisation O/N @ 42C (or at least 16 hrs).

Washes consist of :
	2 x 30 min with : 2XSSC + 0.1% SDS @ RT followed by
	2 x 30 min with : 0.2XSSC + 0.1% SDS @ 55C.

Air dry before covering in wrap and apposing to either phosphor imaging 
plates or X-ray film.
Note:  it is best to check the membrane before apposing as if the probe 
is too hot you can do an additional wash.

Hope this is some help

Regards

Patrick HJ Falckh PhD
Key Centre for Applied & Nutritional Toxicology
RMIT University - City Campus
Victoria Australia

p.falckh at rmit.edu.au





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