Polymerase III In Vitro Transcription

Description
This protocol describes an RNA Polymerase III (Pol III) transcription assay using an extract or proteins of choice. Pol III is the polymerase responsible for transcribing 5S RNA, tRNAs, and other small RNAs. á-Amanitin inhibits Pol II transcription in the assay. The newly-transcribed, radiolabeled RNA is visualized by autoradiography following Urea Polyacrylamide gel electrophoresis.

Procedure
A. RNA Polymerase Reaction (see Hint #1)

1. Prepare a dNTP mix containing:

2 ìl of dNTP Stock

0.5 ìl of á-[32P]-GTP, 3,000 Ci/mmole (CAUTION! see Hint #2)

Add an appropriate amount of DNA template

Add ddH2O to a final volume of 3 ìl

2. Prepare enough Transcription Mixture that contains all of the other transcription components except protein for all the reactions to be carried out. The volumes required for a single reaction are as follows:

4 ìl of 5X Pol III Transcription Buffer

0.2 ìl of 0.1 M DTT

0.2 ìl of á-Amanitin

20 Units of RNAguard RNase Inhibitor (Amersham)

Add ddH2O to a final volume of 16 ìl (minus the volume of protein to be added).

3. Aliquot the Transcription Mixture (prepared in Step #2) into microcentrifuge tubes on ice. Prepare enough tubes for all the transcription reactions to be carried out.

4. Add the whole cell protein extract (typically 20 to 40 ìg of protein from a whole cell extract) and any other proteins to be added to the tubes on ice and bring all tubes to the same volume with Extract Dialysis Buffer (or Protein Dilution Buffer).

5. Add 3 ìl of the NTP mix (prepared in Step #1) to each reaction, mix by flicking the tube 5 to 6 times.

6. Immediately incubate at 30°C for 30 min (see Hint #3).

7. Stop the reaction by adding 180 ìl of the Stop Mix.

8. Add an equal volume of Phenol/Chloroform and mix by vortexing.

9. Centrifuge the tubes for 3 min at maximum speed in a microcentrifuge to separate the phases and recover the upper, aqueous phase to a fresh tube.

11. Precipitate the RNA by adding 20 ìl of 3.0 M Sodium Acetate and 600 ìl of 100% Ethanol and incubating at -20°C for 10 min.

12. Centrifuge at maximum speed in a microcentrifuge for 10 min and remove the supernatant with a drawn-out Pasteur pipette.

13. Wash the RNA pellet with 200 ìl of 100% Ethanol.

14. Centrifuge again for 3 min at maximum speed in a microcentrifuge tube and remove the supernatant with a drawn-out Pasteur pipette.

15. Allow the RNA pellet to dry, but do not over-dry. Thoroughly resuspend the RNA pellet in a convenient volume of Formamide Dye Solution (approximately 8 ìl).

16. Heat the RNA samples for 1 min at 90°C and place immediately on ice.

B. Polyacrylamide Gel Analysis

1. Prepare a 6% Urea Polyacrylamide Gel Solution as described in the Solutions Section.

2. Remove 1 ml of the 6% Urea Polyacrylamide Gel Solution and add 1 ìl of TEMED (N,N,N',N'-Tetramethylethylethylenediamine) to it. Use this mixture to pour a polyacrylamide plug for the bottom of a 4 mm thick gel.

3. Allow the plug to solidify for a couple of minutes.

4. Add 18 of ìl TEMED to the remainder of the 6% Urea Polyacrylamide Gel Solution and pour the gel.

5. After it has solidified, clean the wells of the gel to remove excess Urea by rinsing with 1X TBE Buffer.

6. Load the transcription samples.

7. Electrophoresis the gel in 1X TBE at 300 V for 1 hr or until the dye front is three-fourths of the way to the bottom of the gel.

8. Dry the gel for 20 to 30 min in a gel dryer.

9. Expose to X-ray film for 3 hr to overnight (do not use an intensifying screen).

Recipes
dNTP Stock Store at -70°C
5 mM dCTP
5 mM dATP
5 mM dUTP
0.5 mM dGTP

Phenol/Chloroform 2:1 Phenol:Chloroform (CAUTION! see Hint #4)
Store at 4°C in a dark, glass bottle

Pol III Transcription Buffer (5X) 25 mM MgCl2
Store at -20°C
10% (v/v) Glycerol
5 mM EDTA
400 mM KCl
100 mM HEPES, pH 7.9

TBE Buffer (10X) 890 mM Boric Acid
20 mM EDTA, pH 8.0
890 mM Tris

10% Ammonium Persulfate Stable for up to 3 weeks
Store at 4°C
10% (w/v) Ammonium Persulfate

6% Urea Polyacrylamide Gel Solution 8 ml ddH2O
Degas the solution
The final volume should be 20 ml
8.5 g Urea
2 ml 10X TBE Buffer
3 ml of 40% Acrylamide Buffer
Add 175 ìl of 10% Ammonium Persulfate
Microwave for approximately 10 sec to dissolve the Acrylamide

40% Acrylamide Buffer 38% (w/v) Acrylamide (CAUTION see Hint #4)
2% (w/v) Bisacrylamide (CAUTION see Hint #4)

Protein Dilution Buffer Store at -70°C
1 mM DTT
150 mM KCl
1 mM EDTA
10% (v/v) Glycerol
50 ìg/ml Bovine Serum Albumin
20 mM Tris, pH 8.0

Extract Dialysis Buffer 100 mM KCl
Store at -70°C
2 mM DTT
5 mM MgCl2
1 mM EDTA
20% (v/v) Glycerol
20 mM HEPES, pH 7.9

Stop Mix 0.5% (w/v) SDS
Prepare just before use
5 ìg/ml tRNA
10 mM EDTA
0.1 M Sodium Acetate

DEPC ddH2O Autoclave for 15 minutes to inactivate the DEPC (CAUTION! see Hint#4)
Add DEPC to ddH2O to a final concentration of 0.1% (v/v)
Allow solution to sit for at least 12 hours.
Shake up or stir solution.

á-Amanitin 1 mg/ml á-Amanitin

Formamide Dye Solution 0.05% (w/v) Xylene Cyanol FF
20 mM EDTA
0.1 % (w/v) SDS
Prepare in deionized Formamide (CAUTION! see Hint #4)
0.05 % (w/v) Bromophenol Blue

0.1 M DTT

3.0 M Sodium Acetate

Supplies

Tips
1. To minimize degradation of RNA by RNAses, wear gloves when handling samples and reagents and change gloves regularly while working. Treat water and solutions with DEPC (Diethyl Pyrocarbonate) to inactive RNAses and use solutions prepared with RNase-free water and equipment. For more information see Scientific Reference Pages under Working With RNA.

2. CAUTION! When using radioisotopes, always follow your institution's policies and regulations regarding the use and disposal of radioactive materials. Be sure to use proper shielding and take all necessary precautions to protect yourself from undue exposure to radioactivity.

3. The reaction works well in the temperature range of room temperature to 32°C.

4. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.
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