Reverse Transcriptase Assay Using DEAE-Cellulose

Monday, October 20, 2003

Description
Reverse Transcriptase Assay Using DEAE-Cellulose

Procedure
1. Take 10 l 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 l of Tris, pH 8.0

68 l of 0.1 M Magnesium Acetate

2.7 l of 1 M DTT

2.7 l of 10% NP-40

8.3 l of 5 M NaCl

6.8 l of poly-rC (10 mg/ml)

6.8 l of oligo dG (1 mg/ml)

2.5 l of 10 Ci/l [32P]-dGTP

1 to 2 l of 3 mM dGTP

and bring the final volume of the cocktail to 680 l with ddH2O.

Add 25 l of cocktail to the round bottom 96-well plate wells.

3. Incubate at 37C for 4 to 16 hr.

4. Take 10 l 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)

Recipes
10 mg/ml poly-rC

5 M NaCl

10% (v/v) NP-40

1 M DTT

0.1 M Magnesium Acetate

95% (v/v) Ethanol

SSC (20X) pH 7.2
3 M NaCl
0.3 M Sodium Citrate

3 mM dGTP

[32P]-dGTP 10 Ci/ l [32P]-dGTP (CAUTION! see Hint #1)

1 mg/ml oligo dG

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Reverse Transcriptase Assay Using DEAE-Cellulose