T7 RNA Polymerase Purification

Contents

1. Buffers
2. Expression Protocol
3. Purification Protocol
4. RNAse Contamination Assay

1. Buffers

Lysis Buffer:

• 50 mM Tris pH 8.0
• 100 mM NaCl
• 5% glycerol
• 5 mM BME
• 1 mM imidazole

Wash Buffer:

• 50 mM Tris pH 8.0
• 100 mM NaCl
• 5% glycerol
• 5 mM BME
• 10 mM imidazole

Elution Buffer:

• 50 mM Tris pH 8.0
• 100 mM NaCl
• 5% glycerol
• 5 mM BME
• 500 mM imidazole

Storage/Dialysis Buffer:

• 20 mM potassium phosphate pH 7.5
• 100 mM NaCl
• 50% glycerol
• 10 mM DTT
• 0.1 mM EDTA
• 0.2% NaN₃

2. Expression Protocol

1. Transform plasmid into BL21 cells and plate on LB/Carb plates. Grow overnight at 37°C.
2. Pick a single colony and put into 100 mL LB/Carb starter culture. Shake/incubate at 37°C.
3. Inoculate 1L LB/Carb with 20 mL starter culture after it becomes cloudy. Shake/incubate at 37°C.
4. Induce with 500 µL 1M IPTG when the OD₆₀₀ is around 0.5.
5. Incubate overnight at 20°C.
6. Spin down the cells for 20 minutes at 6000 g, 4°C.
7. Store the pellet at -20°C.

Danger

Overnight starter cultures may not express well. Cell pellets were a greenish-black color. Starter culture may take several hours. This plasmid expresses very well!

3. Purification Protocol

1. Resuspend the pellet in 25 mL lysis buffer. Add 10 mg lysozyme, 55 µL 100 mM PMSF, and HALT protease. Nutate 20 minutes at 4°C.
2. Add 250 µL 8% sodium deoxycholate. Nutate 20 minutes at 4°C.
3. Sonicate with microtip at 40% power, 15 sec on, 15 sec off, for a total of 90 seconds on time (so 6 cycles).
4. Centrifuge for 30 minutes at 18000 g and 4°C. Pass supernatant through low protein binding 0.22 µm filters. Save for the column.
5. Pour 5 mL NiNTA agarose into container to settle, and wash with lysis buffer. Repeat. Pour 50% slurry into column and equilibrate with 10 CV lysis buffer. Run column in cold room.
6. Flow supernatant over column and collect flow through.
7. Wash column with 3 CV lysis buffer.
8. Wash column with 10 CV wash buffer.
9. Elute with 10 CV elution buffer. Collect 1 CV fractions.
10. Assay fractions with 8% SDS-PAGE gels. Pool and dialyze fractions into storage buffer at 4°C. Change buffer at least once. You can dialyze overnight.
11. Check protein concentration. A₂₈₀/1.3 gives the concentration in mg/mL.
12. Aliquot and store at -20°C or -80°C.

4. RNAse Contamination Assay

• We need to assay the T7 RNAP for contamination with ribonucleases. No specific protocol was given.