PREHYBRIDIZATION AND PROBE LABELING
- Set hybridization oven to 65°C.
- Thaw 32P isotope in hood.
- Place membrane in hybridization tube.
- Add 50 mL of prehybridization buffer to each tube.
Prehybridization buffer [for 4 probes] ddH20 125 mL 20X SSPE 60 mL 100X Denhardt's solution 10 mL 20% SDS 5 mL
Salmon sperm DNA [10 mg/mL; boil for 5 minutes and set on ice 5 minutes before adding]
1 mL TOTAL 200 mL
- Prehybridize membranes for at least 6 hours to overnight.
- Heat water to boiling in a beaker on a hot plate.
- Labeling components are
Labeling components DNA 1.0 µL [20–50 ng] ddH20 4.0 µL [total volume of H20 and probe = 5 µL] Oligo primers 1.0 µL dNTPs [5 mM] 1.5µL
1.0 µL 10X Klenow buffer 1.5µL dCTP 32P 5.0 µL TOTAL 15.0 µL
- Conbine ddH20, oligo primer, and DNA in a 1.5-ml microfuge tube, boil for 4 minutes, and place on ice.
- Add dNTPs, 10X buffer, Klenow enzyme, and 32P. Mix, spin briefly, and allow reaction to go overnight at room temperature or place at 37°C for 2 hours.
- Add glass wool to the base of a 1-mL syringe and place in a 15-mL centrifuge tube.
- Using a transfer pipet, fill the syringe with a solution of Sephadex G-50 in TE. Avoid getting air bubbles in the column.
- Centrifuge tubes for ~30 seconds, refill tubes with fresh Sephadex G-50 solution, and centrifuge at 1,500 RPM for 4 minutes.
- Add 200 µL of TE to each column and centrifuge for 4 minutes at 1,500 RPM.
- Add 185 µL of TE to each probe.
- Cut the cap off the tube and withdraw all of the probe solution from the tube with a pipet. Place an empty tube under the syringe and add the probe to the top of the column.
- Spin the column for 4 minutes at 1,500 RPM.
- Discard the column; remove tube with pruified probe and recap.
- Add a cap-lock to the tube, boil for 4 minutes, and place on ice.