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PCR Method to Make Radioactive Probes

来源:生物谷  2010-7-2   访问量:1503
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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 (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
      _________________
      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.

Southern Hybridization
    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.

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