Probing of Northern blot
1. Label the membranes in order to recognize orientation and RNA side. They can eventually be cut. Crosslink in the Stratalinke (”auto crosslink”; RNA side up). Bake membranes at 80℃ for 30´.
2. Prewet the membrane in H2O (remove surplus H2O). Prehybridize for 1 hour at 65 ℃ in Quikhyb (use 15 ml for the medium and 20 ml for the big hybaid tubes).
3. The labelled probe is denatured at 95℃ for 5´ and put on ice. Transfer to 2 ml prevarmed Quikhyb buffer (65℃), mix and immediately add the mixture to the prehybridized membrane (or distribute between several membranes).
Wash blot
15 min in 2X SSC + 0.1% SDS at room temp
2x 15min in 0.2X SSC + 0.1% SDS at 65℃
Read blot on phosphoimager screen
Notes:
Rinse gel chambers and combs in 50mM NaOH for 1 hour
Strip blot by adding boiling water and cool.
Solutions:
10X MOPS/EDTA:
0.5 M MOPS (104.65 g)
0.01 M EDTA (20 ml 0.5 M EDTA)
H2O to 900 ml
pH to 7.0 with NaOH
H2O to 1000ml
(0.1% DEPC ~1ml to 1L)
Sample buffer, (make fresh):
196μl deionised formamide
39μl 10X MOPS/EDTA
63μl formaldehyde (37%)
36μl 50% glycerol
BPB ~16μl (brom phenol blue)
350 ml total
Northern Gel:
Dissolve in the microwave oven. Transfer to fume hood and add formaldehyde. Pour the gel and let sit for 1 hour in hood. If the gel is not used after 1 hr, add water to the gel surface to avoid drying out.
Gel treatment: 50mM NaOH, 2g
150mM NaCl, 8.77g
H2O to 1000 ml
100mM Tris, pH7,5. 12.11g
150mM NaCl, 8.77g
H2O to 1000 ml
DEPC water: 0.1% DEPC (Diethyl pyrocarbonat 97%)
ddH2O -> autoclave
