Hybridisation buffer: 80%Deionised formamide, 40mM PIPES pH6.7, 400mM NaCl, 1mM EDTA
RNase digestion buffer: 10mM Tris pH 7.5, 5mM EDTA, 300mM NaCl.
400mM PIPES pH 6.7, 10mM EDTA.
4M NaCl
RNase solution: 40 ug/ml RNase A, 2ug/ml RNase T, (in RNase digestion buffer).
Proteinase K solution: 20mg/ml. (!!!Aliquot in small amounts DO NOT freeze thaw!!!)
Loading buffer: 7M Urea, 1 x TBE, 0.1% Bromophenol Blue, 0.1% Xylene Cyanol.
1. Dissolve hot probe (~2-7 x10exp5 cpm per reaction) in hybridisation buffer. Heat at 100 oC 5' cool on ice.
2. Mix as follows.
Formamide: 24ul
400mM PIPES, 10mM EDTA: 3ul
4M NaCl 3ul
RNA 1-2ul (~5ug/reaction)Probe 1ul (2-7x10exp5 cpm)
3. Heat to 85 oC then incubate o/n at 45 oC.
4. Add 300ul RNase solution, incubate at 30 oC.
5. Add 20ul 10% SDS, 2.5ul proteinase K (20mg/ml). Incubate 10' at 45 oC.
6. Extract once with phenol/chloroform (you can increase the volume first).
7. Add 20ug tRNA (carrier) and precipitate with EtOH.
8. Wash pellet with 80% EtOH twice, speedvac dry and resuspend in loading buffer.
9. Denature sample at ~90 oC 5'.
10. Run on 6% acrylamide gel.
11. Fix, dry and autoradiograph.
12. Publish paper.