| Day | Activity |
| -1 | 05:00pm: Transform RIL cells |
| 0 |
09:00am: Retrieve plates and put in fridge 05:30pm: Autoclave 8L LB 06:00pm: Overnight 250ml culture + kanamycin |
| 1 |
08:00am: Retrieve 8L LB flasks and leave to cool 09:00am: Innoculate each flask with 25ml of overnight culture + kanamycin, put in incubator 10:00pm: Prepare buffers for the week, pour two gels: one 10-lane for expression study, one for tommorow 12:00pm: Take OD600, induce if ready 05:00pm: Take samples and run on a gel; spin down cultures in centrifuge, discard supernatants and store pellets overnight in freezer Make buffers: Inclusion body prep and refolding step. |
| 2 |
10:00am: Inclusion body preps, leave overnight to solubilise |
| 3 |
09:00am: Recover protein & spin down at 45000g. Separate out supernatant and spin again. Repeat as necessary. 10:00am (if you're lucky!): Decant protein, incubate 1hr with 5mM DTT. 11:00am: Filter protein and prime Akta. 12:00pm: Refold on Akta. Meanwhile, make buffers for next stage. 02:00pm: Dialyse protein 04:00pm: Change dialysis buffer 06:00pm: change dialysis buffer, leave overnight Or 02:00pm: Concentrate sample, run desalting column (can do TEV digest overnight at 4deg at this stage) on Akta, then dilute again. Obviously not ideal due to concentrating followed by re-diluting. |
| 4 |
08:00am: Recover protein 09:00am: Do TEV digest 12:00pm: Concentrate protein 02:00pm: Add GdnHCl and NaCl to protein 03:00pm: Run protein on nickel column on Akta 04:00pm: Collect flowthrough and dialyse 04:30pm: Start to equilibrate Superdex 200 on Akta 06:00pm: Change dialysis buffer 08:30pm: Change dialysis buffer run gels |
| 5 |
Size exclusion on Akta Recover protein, concentrate as necessary Set Superdex 200 to store using Akta |