NORTHERN BLOT AND HYBRIDIZATION

Run a  1.5 - 1.8 % agarose formaldehyde gel.

Blot directly into Nytran using 10 x  SSC , overnight at room temperature.
Remove membrane (trim if necessary) and put in a clean glass plate having the face in contact with the gel up.
Cover with Saran Wrap and remove air bubbles that may be trapped.

UV cross link.
Remove membrane from plate and put in 1 x SSC - 0.1 % SDS  at 50 o C for 45 minutes.

To probe with random primed DNA 

Put membrane in HMA -50

5 x SSC
0.2% SDS, 
0.02% BSA
0.02% Ficoll
0.02% PVP
0.5 mg / ml sonicated, denatured, Salmon sperm DNA
50 % formamide

Keep solution at 4 o C

Use to pre-hybridization  and hybridization.
37 o C overnight.

Washing: 0.1 x SSC , 0.1%SDS at 42 o C . 
4 x  20 to 30 minutes each washing.

To probe with oligomers

Put membrane after UV and washing in :
6 x SSC
5 x Denhardts
0.5 mg / ml sonicated , denatured, Salmon sperm DNA
37 oC overnight
Use to pre-hybridize and hybridize .

Wash filters using the Tetramethylammonium chloride protocol (PNAS , 1985 vol. 82, 1585-1588)