PCR Method to Make Radioactive Probes

Materials

Method

1. Combine reaction mix on ice:
   
   16.6 µl 30% Glycerol
   16 µl 100 mM (NH₄)₂SO₄
   6.8 µl 1 M Tris, pH 8.5
   2.5 µl 100 mM MgAc₂
   1 µl 1% Triton X-100
   0.8 µl hot probe dNTP mix
   40 pmols of each primer
   0.5 to 1 µg of template
   0.4 µl Taq polymerase (5 Units/µl)
   ____________________________
   ==> H₂O to 95 µl
   Then add 5 µl α-³²P dCTP (3000Ci/mMole)
   
   
2. PCR cycle profile:
   
   94°C 5 minutes
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   94°C 30 sec
   55°C 30 sec
   72°C 30 sec
   ==> 10 cycles
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   72°C 7 minutes
   
   
3. Clean probe on Sephadex G50 spin columns.
4. Check activity with scintilation counter.

1. After transfer, crosslink blot and hybridize for 5 min in hybridization solution at 42°C.
   
   
2. Boil probe for 5 minutes in 10 to 20 ml hyb solution.
   
   
3. Pour off hyb solution from blot and add probe. Incubate overnight at 42°C.
   
   
4. Pour off probe and wash blot 2X 5 minutes in the hyb tube with Blot Wash 1.
   
   
5. Remove blot from bottle and wash 2X 15 minutes at 55°C with Blot Wash 2.
   
   
6. Check radioactivity associated with corner of blot. If still hot: 15 min 55°C Blot Wash 2.
   
   
7. Wrap blot in plastic wrap and put down on phosphorimager cassette or film.