PCR Southern blot hybridization using oligonucleotide probe

PCR Southern blot hybridization using oligonucleotide probe:

1. Capillary transfer DNA (PCR product) on nylone membrane overnight.
2. Neutralize membrane in 0.5M Tris.HCl + 1.5M NaCl for 30 min.
3. Dry membrane.
4. Fixation under UV-lamp. (less than 1min)
5. Wash membrane with 6x SSC briefly (2min).
6. Wash membrane in 6x SSC + 0.5% SDS for 30 min.
7. Incubate membrane in hybridization buffer: 6x SSC + 0.5% SDS + 5x
Denhardt buffer at 50c for 30 min.
8. Boil to denature haringsperma DNA (10mg/ml) for 5 min. Snap chill on ice.
9. Prehybridization: 25 ml hybridization buffer + 400 ul haringsperma
DNA at 50c for 30 min.
10. Add labeled oligonucleotide probe.
11. Perform Hybridization at 50c (water bath or incubator) overnight
with gentle shaking or rolling.
12. Wash membrane in 200ml 2x SSC + 0.1% SDS at room temperature for 1 hour.
13. Wash membrane in 200ml 2x SSC for 30 min, change fresh 2x SSC for
another 30 min.
14. Expose membrane to x-film for 1-2 days @ -70c.
15. Prewarm cassette in room temperature for 30 min before film development.

Last update 29-Aug-2006, Rating Good of 1 votes.