- Put the filter to be hybridized to in a seal-a-meal bag, & add 5-10ml (10ml, unless the filter is small) of hybridization sol'n. Squeeze out all the bubbles & seal. Sometimes it helps to "roll" out the bubbles with a pipette. Be SURE that the bag is well sealed & won't leak!
- Incubate at 65C for 1-2hr. This is best done in a water bath, & if the filter is vertically oriented the bubbles will float to the top, away from the filter. No shaking is required.
- Cut off a corner of the bag & add the radio-labeled probe. If the probe is double-stranded DNA, preheat the probe in a boiling water bath for 2 min., & quick chill on ice before adding to the bag. Hybridize at 65C overnight.
- Wash twice for 5 min. at RT in 40ml wash sol'n #1 at RT for 5min. each. This is done by cutting off a corner of the bag & draining all of the hybridization sol'n away (use a pipette to roll out the last of the sol'n) & then refilling the bag, via pipette or syringe, with the wash sol'n, & incubating. Check the filter with a geiger-counter; if the filter seems "cold", stop at this point & put the filter on film (see below, step 8).
- Wash with 20ml Wash sol'n #2 at 65C for 15 min.
- Wash with 20ml Wash sol'n #3 at 65C for 10 min.
- Drain the last wash sol'n, & use a pipette to "roll-out" the last of the fluid, then reseal the bag without trapping air.
- Autoradiograph as usual.