Polymerase III in vitro Transcription
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Polymerase III in vitro Transcription
Steve Hahn
For the following reactions, use appropriate shielding and dispose of radioactive waste properly!
A 20 microliter transcription reaction contains:
4.0 μl 5X Pol III transcription buffer
0.2 μl 0.1 M DTT
0.2 μl alpha amanatin (1 mg/ml)
0.2 μl RNAse Inhibitor (Amersham, 20 U)
2.0 μl NTP stock
0.5 μl alpha 32P GTP
140 ng plasmid template (pSK LEU3, pCH6), [use 280 ng 5S pUC template]
H2O to 20 microliters final volume.
Transcription Protocol:
For a Typical transcription reaction, make an NTP mix containing:
2.0 μl NTP stock
0.5 μl alpha 32P GTP
DNA template
H2O to a final volume of 3 microliters
Make a second mix containing all other transcription components except protein:
4.0 μl 5X Pol III transcription buffer
0.2 μl 0.1 M DTT
0.2 μl alpha amanatin (1 mg/ml)
0.2 μl RNAse Inhibitor
H2O to a final volume of 16 microliters less the maximum volume of protein to be added.
Aliquot the second mix to all reaction tubes and incubate on ice.
Add the protein extract (typically 20-40 micrograms of a whole cell extract) and any other proteins to be added. Add extract dialysis buffer (or protein dilution buffer) to bring all reactions to the same volume.
Add 3 μl of the NTP mix to each reaction, mix by flicking the tube 5-6 times, and immediately incubate at 30 degrees (the reactions will work well between room temp and 32 degrees).
After 30 min., stop the reaction with 180 microliters stop solution.
Extract once with phenol/CHCl3 (2/1). ETOH precipitate with 20 μl 3M NaOAc and 600 μl ETOH.
Freeze 10 min. Spin 10 min. Remove sup. with a drawn out pasteur pipette. Add 200 μl 100% ETOH and spin 3 min. Remove sup. again.
Dry pellets but do not overdry. Thoroughly resuspend pellets in Formamide dye solution containing 0.1% SDS.
Heat 1 min. 90 degrees and put on ice. Load to 0.4 mm thick urea acrylamide gel (don’t forget to clean the wells of the gel just before loading). Typically, run 300V, 60 min until the bromphenyl blue is 3/4 of the way to the bottom.
Dry gel 20-30 min. Typically expose ~3 hr or overnight without a screen.
Solutions needed for Pol III transcription:
5X Pol III Buffer (store frozen) (1 ml):
|
10% glycerol |
200 ul 50% glycerol |
|
100 mM HEPES, 7.9 |
100 ul 1M HEPES |
|
400 mM KCl |
400 ul 1M KCl |
|
25 mM MgCl2 |
25 ul 1 M MgCl2 |
|
5 mM EDTA |
20 ul 0.25 M EDTA |
|
|
255 ul H2O |
NTP Stock (store frozen ―70 deg):
5 mM each ATP, CTP, UTP
0.5 mM GTP
7.3 ml Stop mix (make fresh):
|
0.1 M NaOAc |
250 ul 3M NaOAc |
|
10 mM EDTA |
300 ul 0.25 M EDTA |
|
|
6.4 ml H2O |
|
0.5% SDS |
375 ul 10% SDS |
|
5 ug/ml tRNA |
1.2 ul tRNA (30 ug/ml) |
Extract Dialysis Buffer (store frozen ―70 deg):
20 mM HEPES, 7.9
100 mM KCl
5 mM MgCl2
1 mM EDTA
20% glycerol
2 mM DTT
Protein dilution buffer (5 ml) store -70 deg:
|
20 mM Tris 8.0 |
100 ul 1 M Tris 8.0 |
|
1 mM DTT |
50 ul 0.1 M DTT |
|
10% glycerol |
1 ml 50% glycerol |
|
150 mM KCl |
0.75 ml 1 M KCl |
|
1 mM EDTA |
20 ul 0.25 M EDTA |
|
50 ug/ml BSA |
2.5 ul 100 mg/ml BSA |
|
|
H2O to 5 ml total volume |
20 ml Acrylamide Urea Gel:
3 ml 40/2% sequencing acrylamide
2 ml 10X TBE
8.5 g Urea
8 ml H2O
microwave ~10 sec and stir to dissolve.
Degas
Add 175 μl 10% ammonium persulfate
Remove 1 ml acrylamide and add 1 μl TEMED. Pour plug at bottom of gel. Let polymerize a few minutes.
To the remaining acrylamide, add 18 microliters TEMED and pour gel. Gel can stored O/N, if kept wet and sealed at room temp.
