3.18. Testing a Glycerol Stock for Protein Over-Expression

1. Have at least 10 ml of sterilized LB media available.
2. Transfer 10 ml of LB media to a 15 ml centrifuge tube.
3. Add 10 μl of antibiotics to the centrifuge tube.
4. Mix the contents of the tube by inverting the tube.
5. To each of 5 culture tubes, add 2 ml of media.
6. Inoculate the 2 ml cultures at the end of the day (4:00 p.m. - 5:00 p.m.) with a glycerol stock.
7. Allow the cultures to grow overnight in a shaking incubator at 200-250 rpm and 37°C.
8. The next morning (10:00 a.m.) transfer 1 ml from each culture tube into 5 new culture tubes.
9. Induce the new culture tubes with 1 mM IPTG.
10. Allow the old and new cultures to grow at 200-250 rpm and 37°C for 4-5 hours.
11. Transfer the 1 ml cultures into 10 different 1 ml centrifuge tubes.
12. Centrifuge the cells at 10,000 g for 10 mins.
13. Pour the supernatant into a waste container (a beaker or 50 ml centrifuge tube) to be sterilized later.
14. Freeze the cells at -20°.

Handcasting Polyacrylamide Gels

1. See 4.15 for instructions.

Preparing Cells for Running

1. Prepare part 1 of 5X sample buffer without DTT.
2. Transfer 1 ml of the 5X sample buffer without DTT to a 1 ml centrifuge tube.
3. Prepare part 2 of 5X sample buffer.
4. Combine 450 7 μl of part 1 with 50 μl of 5M DTT.
5. Add 100 μl of 1X sample buffer to pelleted cells.
6. Resuspend cells by vortexing.
7. Heat the cells for 5 mins at 90°C.
8. Freeze the cells at -80°C.
9. Repeat the previous two steps three times.
10. Centrifuge the cells at 10,000 g for 10 mins.

Running Polyacrylamide Gels

1. See 4.16 for instructions.

Staining/Destaining Gels

1. See 4.17 for instructions.