1 Jul 2011


NOTE:   The following protocol assumes that the individual performing the procedure has been
Appropriately trained in the proper handling of radioactive isotopes and will therefore observe the necessary
Precautions and regulations regarding personal/lab safety.
1. After Southern transfer is complete, place the blot in the UV Crosslinker and crosslink on the automatic
2. Prepare prehybridization solution.
A. Warm 10mL/blot of prehybridization solution to 42°C.
B. Denature salmon sperm by heating sample to 95°C for 5 minutes, chilling sample quickly on
Ice, and microcentrifuging the sample. (Stock solution is 20mg/mL;  50mg/mL is needed; so
Add 25-30mL/10mL prehybridization solution.)
C. Add denatured salmon sperm to warmed prehybridization solution.
3. Carefully roll blot and place in hybridization tube.  Add prehybridization solution to the tube.
4. Place tube in 42°C hybridization oven for 5 hours.
5. Make probe buffer, and then probe mix.  After adding the isotope to the probe “mix”, incubate the
Sample in a 37°C water bath for 40 minutes.
6. Separate the nonincorporated label with a G-25 Sephadex spin column.  Determine the specific activity
Of the probe by liquid scintillation. Add 1 mL of the probe to 5 mL of scintillation fluid.
7. Warm the hybridization solution to 49°C.  Add 10-15 x 10^6 Cpm of the probe to 10mL of the
Hybridization solution. Mix the solution.
8. Pour off the prehybridization solution from the blot and add the hybridization solution.
9. Incubate the container at 49°C overnight.
10. Remove the hybridization solution and discard properly.  Preheat the 6X wash solution to 49°C.
11. Cover the blot with wash solution.  Shake the blot covered with 6X wash solution for 15 minutes at
49°C.  Remove the membrane and blot dry with absorbent paper.
12. Preheat the 2X wash solution to 49°C.  Wash the blot as described above for only ~30 sec.
13. Check the blot with a radiation detection device.  The background on blank areas of the membrane
Should be 200 cpm or less. If the background is too high, repeat wash with 2X wash solution.
14. Quantify the blot by the method of your choice.

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