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Radioactive Probes

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 l 30% Glycerol
      16 l 100 mM (NH4)2SO4
      6.8 l 1 M Tris, pH 8.5
      2.5 l 100 mM MgAc2
      1 l 1% Triton X-100
      0.8 l hot probe dNTP mix
      40 pmols of each primer
      0.5 to 1 g of template
      0.4 l Taq polymerase (5 Units/l)
      ____________________________
      ==> H2O to 95 l
      Then add 5 l α-32P dCTP (3000Ci/mMole)

    2. PCR cycle profile:

      94C 5 minutes
      _________________
      94C 30 sec
      55C 30 sec
      72C 30 sec
      ==> 10 cycles
      _________________
      72C 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 42C.

    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 42C.

    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 55C with Blot Wash 2.

    6. Check radioactivity associated with corner of blot. If still hot: 15 min 55C Blot Wash 2.

    7. Wrap blot in plastic wrap and put down on phosphorimager cassette or film.
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