| Contributor: |
The Laboratory of J. Michael Bishop at the University of California, San Francisco
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1. Take 10 μl of medium from the wells of a 96-well microtiter plate containing a confluent cell culture of the desired cell line and put it into a new 96-well round bottom plate.
2. Prepare the reaction cocktail by combining the following:
68 μl of Tris, pH 8.0
68 μl of 0.1 M Magnesium Acetate
2.7 μl of 1 M DTT
2.7 μl of 10% NP-40
8.3 μl of 5 M NaCl
6.8 μl of poly-rC (10 mg/ml)
6.8 μl of oligo dG (1 mg/ml)
2.5 μl of 10 Ci/μl [32P]-dGTP
1 to 2 μl of 3 mM dGTP
and bring the final volume of the cocktail to 680 μl with ddH2O.
Add 25 μl of cocktail to the round bottom 96-well plate wells.
3. Incubate at 37°C for 4 to 16 hr.
4. Take 10 μl from each well and spot it onto a DEAE cellulose filter.
5. Rinse the filter 2 to 3 times with 50 ml of 2X SSC and then rinse it twice with 50 ml of 95% Ethanol.
6. Air-dry the DEAE-cellulose filter (see Hint #2).
7. Autoradiograph the filter between 4 and 16 hr (see Protocol on Autoradiography)
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| [32P]-dGTP |
| 10 Ci/μl [32P]-dGTP (CAUTION! see Hint #1)
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| 1 mg/ml oligo dG |
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| 10 mg/ml poly-rC |
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| 5 M NaCl |
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| 10% (v/v) NP-40 |
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| 1 M DTT |
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| 0.1 M Magnesium Acetate |
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| 95% (v/v) Ethanol |
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| SSC (20X) |
| pH 7.2
3 M NaCl
0.3 M Sodium Citrate
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| 3 mM dGTP |
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Poly-rC
Sodium Citrate
Oligo dG
[32P]-dGTP
Tris
Sodium Chloride
Magnesium Acetate
dGTP
DTT
NP-40
Ethanol
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1. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.
2. You can speed up this air-dry step with a heating lamp.
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