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- 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)
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==> H2O to 95 µl
Then add 5 µl α-32P dCTP (3000Ci/mMole)
- PCR cycle profile:
94℃ 5 minutes
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94℃ 30 sec
55℃ 30 sec
72℃ 30 sec
==> 10 cycles
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72℃ 7 minutes
- Clean probe on Sephadex G50 spin columns.
- Check activity with scintilation counter.
- After transfer, crosslink blot and hybridize for 5 min in hybridization solution at 42℃.
- Boil probe for 5 minutes in 10 to 20 ml hyb solution.
- Pour off hyb solution from blot and add probe. Incubate overnight at 42℃.
- Pour off probe and wash blot 2X 5 minutes in the hyb tube with Blot Wash 1.
- Remove blot from bottle and wash 2X 15 minutes at 55℃ with Blot Wash 2.
- Check radioactivity associated with corner of blot. If still hot: 15 min 55℃ Blot Wash 2.
- Wrap blot in plastic wrap and put down on phosphorimager cassette or film.
