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.
- Abigail E. Miller 20:19, 7 October 2012 (EDT):what time did this occur at? since the amount of time affects the amount of cell growth, recording hte time things occur is important.
- 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.
- The cell cultures continued to shake in the incubating orbital shaker for 3-4 hours.
- Abigail E. Miller 20:19, 7 October 2012 (EDT):what time? be specific in your notebook. referencing 3-4 hours is a general procedure, not exactly what you did.
- The cells were harvested by centrifuging them at 4500rpm for 15 minutes.
- The cells were collected and placed in the -80°C freezer.
- Abigail E. Miller 20:19, 7 October 2012 (EDT):how were the cells collected? they should have been separated form the supernatant nad the cells in hte pellet resuspended in the binding buffer (x mM Tris, y mM NaCl and z mM imidizole)
- 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.)
- Abigail E. Miller 20:20, 7 October 2012 (EDT):what are the theoretical and actual concentrations?