PCR Method to Make Radioactive Probes

Materials

Hot Probe dNTP Mix:
25 mM dATP
25 mM dTTP
25 mM dGTP
2.5 mM dCTP

Hybridization Solution:
5X SSC
0.5% (w/v) Blocking Reagent
0.1% (w/v) N-lauroylsarcosine, Na-salt
0.02% (w/v) SDS
50% Formamide

Blot Wash 1:
2X SSC
0.1% SDS

Blot Wash 2:
0.1X SSC
0.1% SDS

Method

PCR Reaction

1. Combine reaction mix on ice:
   
   16.6 µl 30% Glycerol
   16 µl 100 mM (NH ₄ ) ₂ SO ₄
   6.8 µl 1 M Tris , pH 8.5
   2.5 µl 100 mM MgAc ₂
   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)
   ____________________________
   ==> H ₂ O to 95 µl
   Then add 5 µl α- ³² P dCTP (3000Ci/mMole)
    
2. PCR cycle profile:
   
   94℃ 5 minutes
   _________________
   94℃ 30 sec
   55℃ 30 sec
   72℃ 30 sec
   ==> 10 cycles
   _________________
   72℃ 7 minutes
    
3. Clean probe on Sephadex G50 spin columns.
4. Check activity with scintilation counter.

Southern Hybridization

1. After transfer, crosslink blot and hybridize for 5 min in hybridization solution at 42℃.
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℃.
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℃ with Blot Wash 2.
6. Check radioactivity associated with corner of blot. If still hot: 15 min 55℃ Blot Wash 2.
7. Wrap blot in plastic wrap and put down on phosphorimager cassette or film.