Southern Hybridazation
Hybridization
Materials
| Prehybridization Solution | (10 - 50 mL). Mix by rotating at 37°C: |
| Start with 50 mL Stark's Solution (with 50% formamide) | |
| + 0.5% non-fat dry milk (0.25 gm) | |
| + 1% SDS (0.5 gm) | |
| + 8% dextran sulfate (4 gm, Sigma D-6001) | |
| 42°C shaking water bath | |
| 100°C Heater block | |
| 32P-labled probe | use 10 - 20 ng DNA/filter, or about 10 million CPM/filter for Southern's |
| Nylon filter | DNA previously blotted onto filter (e.g. GeneScreen+) |
| Hybridization Chamber | use Glass bell jar for 9 cm disk filters |
| or Seal A Meal bag and glass tray for larger filters | |
Procedure
1) Mix Prehybridization Solution until dissolved. Incubate filters at 42°C in Prehyb solution in either the glass bell jar or a Seal A Meal bag for 1-4 hrs.
2) Turn on heater block to 100°C.
3) Transfer probe to 1.5 mL eppendorf tube with a pinhole in the cap and add H2O to bring the total volume to 150 µL. Denature the DNA by heating to 100°C for 5 min.
4) Transfer probe to the filter and chamber. Mix well with the Prehyb solution. Reseal the bag or put a lid and weight on the bell jar. Incubate with shaking at 42°C overnight.
Washing Filters
Materials
1) Discard the radioactive Hybridization solution into absorbed liquid waste container. Rinse the filter with 1x SSC, 0.1%SDS and discard.
2) Wash the filter in 1x SSC, 0.1% SDS in a shaking water bath for 1 hour. Meanwhile allow the water bath to heat to 65°C. Change wash solution 2x during the hour.
3) Wash the filter in 0.1x SSC, 0.1% SDS at 65°C for 10 to 20 minutes. Meanwhile monitor the CPM on the filter with a Geiger counter. Stop washing the blot when the counts are reduced to 2 - 4x background.
4) Blot the filters on Whatman 3MM paper. Wrap the filter in Saran wrap and place glow in the dark stickers for orientation. In a dark room place the filter in an autoradiograph casette then a piece of X-ray film, and then an intensifying screen. If a weak signal is anticipated do this in complete darkness (with the safe lights off).
5) Place the film casette at -70°C. For cloned DNA and high S.A. probes expose for 2 - 4 hours. For mammalian genomic DNA Southern blots expose the film for 18 - 72 hrs.
6) Remove the film casette and allow it to warm for 30 min. Remove the film in the dark room (for weak signals do this with the safe lights off) and develop in a processor or in developing tanks as follows: X-ray developer: 2 min. Water wash 10 sec. Fixer: 2 min. Water wash 5 min. Air dry the film.
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