Shock--nothing at all after washing membrane in northern blotting - (Apr/29/2007 )
Followed the procedure of mRNA detection, every stage was right and showed a good result before the wash stage. Before this stage, we also detected whether RNA was transferred to the membrane and hybridization was right, they both showed OK. The washing details show as below:
1. Remove the membrane from the hybridisation bottle using tweezers and float into the first solution in a small container.
2. Wash membrane 2x for 5 min at RT in 2x SSC (0.1 % SDS).
3. Wash membrane 15 min at 65C in 0.5x SSC (0.1 % SDS). Do this by adding to the hyb bottle before placing in the hyb oven.
4. Then wash for 10 min at 65C in 0.2x SSC (0.1 % SDS)
Detection of RNA species
1. Equilibrate membrane in buffer 1 for 2 min.
2. Rinse the hybridisation bottle thoroughly to remove SSC-SDS.
3. Add 10 mL of buffer 2 to the hybridisation bottle, put membrane back in and remove all air bubbles. Make sure you ask the demonstrator if having issues.
4. Incubate for 30 min (or longer) at RT
5. Add 1 L of anti-DIG antibody per 10 mL of buffer 2 (prior to use spin antibody for 5 min at 4C)
6. Incubate for 30 min (NOT longer) at RT
7. Pour off solution and wash 2x 15 min in buffer 1 in the hyb bottle.
8. Remove membrane, place in a dish containing buffer 3 and incubate for at least 2 min.
9. Dilute CSPD (chemiluminescent substrate) 1:100 in buffer 3 (1000 L required for 10 x 10 cm membrane - 10L CSPD in 990L buffer 3 in an Eppendorf tube).
10. Place the membrane, RNA side up, onto open plastic bag (DO NOT LET MEMBRANE DRY!)
11. Pipette CSPD onto membrane immediately, scattering drops over the surface
12. Cover with top piece of plastic, avoiding any air bubbles
13. Incubate for 5 min at RT.
14. Remove residual CSPD(by squeezing and blotting with a piece of tissue as demonstrated), seal plastic bag and incubate membrane at 37C for 15 min
15. Place membrane into a cassette and expose to X-ray film overnight at RT.
But we donot detect why the result just showed a blank paper. Could anyone propose some reasons about that, please?
Thank you very much!
1. RNAse in one of your wash buffers.
2. Too high a stringency in your wash. Try leaving out the 0.2x SSC wash, or lowering the wash temperature.
You can debug this by using serial dilutions of your sample spotted on membranes, rather than a blotted gel. This will save you a lot of time and grief.
Your suggestion and analysis is meaningful, I will try it later. Because this experiment has been done last year, the result showed pretty good.
Maybe subtle conditions were changed also.
Anyway, thanks for your suggestions.