In the morning (~8:30am)
- to prepare the cells for protein expression and
- to make IPTG
In the afternoon (~12:15pm)
- to finish preparing the cells for protein expression and
- to make binding and elusion buffers.
Prepared in the Morning
*Preparing the Cells for Protein Expression
- The starter cultures were centrifuged at 4500rpm for 15 minutes.
- The cell pellets were resuspended in 4mL of fresh LB (from one of the Fernbach flasks)
- The expression culture media was inoculated by dividing the resuspended cells among the Fernbach flasks.
- The aluminum covering the Fernbach flasks were re-sterilized before recovering the flasks.
- The expression cultures were incubated at 37°C and 160rpm at 9:00AM.
- 0.4 g of IPTG was added to 4mL to create 0.4M IPTG solution.
Prepared in the Afternoon
*Preparing the Cells for Protein Expression (continued)
- 1mL of 0.4M IPTG was added to each flask in order to induce protein expression at 12:15.
- The cell cultures continued to shake in the incubating orbital shaker for 4 hours.
- The cells were harvested by centrifuging them at 4500rpm for 15 minutes and then the supernatant was removed from the centrifuge tube. The pellet was resuspended in 30mL of binding buffer (the procedure for making the binding buffer containing 0.03M Imidazole, 0.02M Tris, and 0.5M NaCl is below).
- The cells were collected and placed in the -80°C freezer.
- The following calculations were used to prepare the binding and elution buffers.
- 1L of water was added to the Binding buffer
- 500mL of water was added to the elution buffer
- HCl was added drop-wise to the buffers to lower the pH to 7.5.
- (The excess HCl was neutralized with NaOH to pH 7, using pH strips, and poured down the sink drain.)
- Because the actual mass and theoretical mass of the chemicals were almost exactly the same, the actual and theoretical concentrations were the same:
- Binding Buffer: 0.03M Imidazole, 0.02M Tris, and 0.5M NaCl
- Elution Buffer: 0.5 M Imidazole, 0.02M Tris, and 0.5M NaCl