| Contributor: |
The Laboratory of Michael Chamberlin at the University of California, Berkeley
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| See a Protocol for the Electrophoresis of RNA through Agarose-Formaldehyde Gels and see a Protocol for Northern Blotting to obtain instructions on RNA transfer, making the required probe, and probe/filter hybridization. |
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1. Following an overnight hybridization of a filter with a probe, place the filter in 2X SSC (prewarmed) at 37°C for 30 min with gentle shaking.
2. Pour off solution and replace with 100 to 300 ml of Wash Buffer 1 and wash the filter for 15 min at 50°C with gentle shaking. Repeat this wash step one time.
3. Pour off the solution and replace it with 100 to 300 ml of Wash Buffer 2 and wash the filter for 15 min at 50°C with gentle shaking. Repeat this wash step one time.
4. Pour off the solution and replace it with 100 to 300 ml of Wash Buffer 3 and wash filter for 15 min at 50°C with gentle shaking. Repeat this wash step one time.
5. Pour off the solution and replace it with 100 to 300 ml of Wash Buffer 4 and wash the filter for 10 min at 50°C with gentle shaking. Repeat this wash step one time.
6. Dry the filter by placing it under a heat lamp for 5 to 10 min.
7. Autoradiograph the blot.
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| 10% (w/v) SDS |
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| Phosphate Wash Buffer (25X) |
| 0.2 M Sodium Phosphate Dibasic (KH2PO4)
1.5% (w/v) Sodium Pyrophosphate
0.3 M Sodium Phosphate Monobasic (Na2HPO4)
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| SSC (20X) |
| pH 7.2
3 M NaCl
0.3 M Sodium Citrate
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| Wash Buffer 4 |
| 0.25X SSC
0.0125% (w/v) SDS
0.125X Phosphate Wash Buffer
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| Wash Buffer 3 |
| 0.25X Phosphate Wash Buffer
0.025% (w/v) SDS
0.5X SSC
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| Wash Buffer 2 |
| 0.05% (w/v) SDS
1X SSC
0.5X Phosphate Wash Buffer
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| Wash Buffer 1 |
| 0.1% (w/v) SDS
1X Phosphate Wash Buffer
2X SSC
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Oligonucleotide
Sodium Phosphate, Dibasic
Sodium Pyrophosphate
SDS
Sodium Chloride
Sodium Phosphate, Monobasic
Sodium Citrate
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No hints are associated with this bioProtocol
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