Blotting and Hybridization with Sodium Phosphate
Categorized: Bacterial Genetics
- Rinse blot with 2X SSC and air dry
- Place blot into hybridization tube with ~100 ml 0.5% SDS, 0.1 SSC. Prehybridize for 1 hour at 65C.
- Discard prehybridization solution and add hybridization buffer; use approximately 7-10 ml per large hybridization tube.
25 ml 1.0 M NaHPO4 200 µl 0.5 M EDTA 50 ml formamide 7 g SDS H2O to 100 ml
- Prehybridize for ~5 – 10 minutes at 37-42ºC.
- Add denatured and labeled probe.
- Hybridize 16 – 24 hours at 37-42ºC.
- Discard hybridization solution to radioactive waste. Wash the membrane briefly in 2X SSC, 0.1% SDS at 23ºC (room temperature).
- Wash the membrane in ~ 100 ml 0.1X SSC , 0.1% SDS for 15-30 minutes at 37-65ºC.
- Repeat step 8.
- Blot the membrane onto filter paper, wrap in plastic and expose to film.
Strip nylon membrane for reuse:
- Wash the blot twice in 100 ml 0.1X SSC, 0.1% SDS at 95ºC for 30 minutes.
- Wash the blot as in step 1 but at 23ºC.