NORTHERN HYBRIDIZATION

1. Prehybridize membrane at 65C overnight. This might be essential!

2. Denature probe: 5 min 100C in heating block. Final concentration in
hybridization: 10-100 ng/ml
3. Take aliquot of prehyb mix, and add denatured probe to aliquot. Then add aliquot
to rest of prehybmix with membrane. Don't add probe directly to membrane!
4. Hybridize O/N at 65C
5. Wash blot 2x 5 min at 65C with low stringency wash buffer, and 2x 30 min at 65C
with high stringency wash buffer
6. Wash blot 2x 5 min at RT with DIG-buffer. It is essential to wash away all salts from
hybridization
7. Block membrane in Blocking buffer for 30-60 min at RT
8. Incubate 30-60 min with anti-DIG-AP (1:10,000 diluted in Blocking buffer)
9. Wash 2x 15 min at RT with DIG-buffer
10. Equilibrate in AP-buffer
11. Do chemiluminescent detection with CDP-Star

Materials
(Pre)hybridization mixture
3M urea
5x SSC
2 % blocking reagent
0.1 % sarkosyl
0.5 % SDS
200 ug/ml total yeast RNA

Wash buffers (prewarmed at 65C)
(Low stringency):
1x SSC
0.1 % SDS
0.1 % sarkosyl

(high stringency):
0.1x SSC
0.1 % SDS
0.1 % sarkosyl

DIG-buffer
0.1 M Maleic acid pH 7.5
0.15 M NaCl
Treat with DEPC, autoclave
Add Tween-20 to 0.1%

Blocking buffer
DIG-buffer with 5% skim milk

AP-buffer
100 mM Tris pH 9.5 (pH 9 is also OK)
100 mM NaCl Autoclave