PCR Method to Make Radioactive Probes

Materials

Method

1. 1. Combine reaction mix on ice:
      
      16.6 µl 30% Glycerol
      16 µl 100 mM (NH₄ )₂ SO₄
      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)
      ____________________________
      ==> H₂ O to 95 µl
      Then add 5 µl α-³² P dCTP (3000Ci/mMole)
       
   2. PCR cycle profile:
      
      94°C 5 minutes
      _________________
      94°C 30 sec
      55°C 30 sec
      72°C 30 sec
      ==> 10 cycles
      _________________
      72°C 7 minutes
       
   3. Clean probe on Sephadex G50 spin columns.
   4. Check activity with scintilation counter.

1. 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.