*3.7.2 Testing for expression*

*Growing and Handling of Bacterial Cultures*

for 2 min at room temperature.

4°C. Discard supernatant.

12.Resuspend dry DNA with TE.

(long term.)

*3.7.1 Protein expression*

tilted.

actual OD600.

supernatant.

4°C.

2.Spin 5 mL of the overnight LB culture at 6000× g for 10 min at 4°C. Discard

3.Add 250 μL Resuspension Buffer containing RNase A to the cell pellet and

4.Add 250 μL Lysis Buffer. Mix gently by inverting the capped tube until homogeneous. Do not vortex. Incubate the tube at room temperature for 5 min.

5.Add 350 μL Precipitation Buffer. Mix immediately by inverting the tube until homogeneous. Do not vortex. Centrifuge the lysate at 16,000× g for 10 min at

6.Add 2–2.5 volumes 95% or 100% ethanol and 1/10 volume of 3 M Na-acetate (pH 4.8) to the supernatant. Invert the microcentrifuge tube to mix. Let stand

10.Centrifuge solution at high speed (at least 16,000× g) for 5 min at room temp.

13.Store plasmid DNA at 4°C (short term) or store the DNA in aliquots at −20°C

The following protocol is written for proteins expressed under the control of the lac, tac, or T7 promoters. The method as described is a generic protocol that can be expanded to test expression in different strains of *E. coli*, induction temperatures,

concentrations of IPTG, or even in the presence of ligands or cofactors.

1.Transform plasmid into an *E. coli* expression strain following Section 3.2.

3.Grow cells for a few hours at 37°C, shaking at 250 rpm. Make sure the tubes are

4.Monitor the turbidity. Once the culture reaches an OD600 of 0.4–0.6 (takes ~2–4 h, depending on the sample), take out 2 mL of the culture. Measure the

7.Centrifuge solution at high speed (at least 16,000× g) for 15–30 min at

8.Open and invert the tube on a paper towel to drain it out.

9.Wash pellet by adding 500 μL cold 70% ethanol.

**3.7 Testing for soluble protein expression in** *E. coli*

2.Inoculate a liquid LB culture following Section 3.4.

Discard supernatant by pipetting it out of the tube.

11.Dry the pellet by inverting over paper towel for 5–20 min.

resuspend the pellet by pipetting until homogeneous.

**46**

