Northern Blotting
1. Make the following gel. MINIGEL MAXIGEL
1-1.5% agarose 0.5-0.75 gm 2.5-3.75 gm
Running buffer 5.0 ml 10X R.B. 25.0 ml 10X R.B.
Water 36.5 ml 182.5 ml
2. Boil. Cool to 65°C. Add 8.5 ml 42.5 ml Formaldehyde in a chemical fume hood (Malinckrodt)
3. Pour gel in a chemical fume hood.
4. Resuspend RNA (10-20 mg whole RNA or 0.5-2.0 ug poly A+ DNA) in 9 ml or 18 ml of:
Water 1.25 ml
10X R.B. 1.0 ml
Formaldehyde 1.75 ml
Formamide 5.0 ml
5. Vortex and heat to 65° for exactly 5 minutes.
6. Add 1/10th volume 10X loading buffer.
7. Run gel in 1X R.B. for 3-6 hours at 65 volts. Bromophenol blue should run 3/4 down to the bottom of the gel.
8. Wet nylon membrane (Hybond, Amersham) in water.
9. Transfer to 20X SSC.
10. Rinse gel in dd H2O for 2 X 15 minutes with gentle agitation.
11. Set up blot in 20X SSC overnight.
12. Mark wells, remove gel. Do not rinse blot.
13. Air dry, and bake at 80°C in vacuum oven 1 hr.
14. Prehybridization mix (40 ml):
Formamide 10 ml (deionized)
1 M Hepes (pH=7.0) 1.0 ml
20 ml/minigel 50X Denhardt's 2 ml
40 ml/maxigel 20X SSC 3.0 ml
Denatured salmon sperm DNA 0.32 ml (10 mg/ml)
10% SDS 0.2 ml
50% Dextran Sulfate 2 ml
Water 1.5 ml
15. Prehyb 42-45°C 3-4 hrs.
15a. Remove 5 ml prehyb from bag for minigel, 15-20 ml for maxigel.
16. Add 10-25 ng denatured hexaprimed probe (note probe can be whole plasmid - not necessary to isolate insert or fragment).
17. Hybridize 42° overnight.
18. Wash:
1XSSC/0.5% SDS 2 x 10 minutes Room temp
0.1X SSC/0.1% SDS 2 x 30 min. 55o C
19. Autoradiogram 4-24 hours with intensifying screen.
Solutions:
10X RUNNING BUFFER: 200 mM MOPS pH=7.0 (pH with NaAc)
50 mM NaAcetate
10 mM EDTA
If needed (usually not), deionize formamide as in Maniatis and store in 5-10 ml aliquots at -20°C.
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