PSF
•High throughput protein expression in microplates•
Protein expression with 96 clones in a deep well microplate. This protocols uses a rich growth medium and a long growth time to maximise the yield of cells and protein.
- Use 2 ml Deep-96well microtitre plates with square wells.
- Fill with 100 µl 2YT, 2% glucose, 100 µg/ml ampicillin, 15 µg/ml kanamycin
- inoculate cultures with replicator with long steel pins
- (for tomorrow: prewarm SB medium containing KPB, 100 µg/ml ampicillin, 15 µg/ml kanamycin, 20 µg/ml thiamin)
- close Store Block with rubber seal ("Noppenmatte")
- shake o/n at 37°C 320 rpm
- add 900 µl prewarmed SB (temperature of induction)
- close plates with microtitre plate lid (lid permits air exchange)
- shake 1-3 h at temperature of induction (usually 37°C) 320 rpm
- The time depends on the strain and on the amount of cells needed. Up to 3 h for SCS1 and XL1Blue. max. 1-2 h for B834 or BL21.
- add 100 µl 11 mM IPTG in water
- shake 3 h at the respective induction temperature, 25°, 30°C or 37°C, at 320 rpm
- Spin 10 min 3500 rpm
- discard supernatants
- freeze pellets at -80°C
SB
| 1 l/0.95 l | 5 l/4.75 l | |
|---|---|---|
| Bactotryptone | 12 g | 60 g |
| Yeast extract | 24 g | 120 g |
| Glycerol 100% | 4 ml | 20 ml |
make up to 950 ml (4750 ml), autoclave 950 ml, cool, add 50 ml of:
1 litre of 20x KPB=46 g KH2PO4 + 243 g K2HPO4 (Mw=174)
4x SB concentrate
| 1 l | 5 l | |
|---|---|---|
| Bactotryptone | 48 g | 240 g |
| Yeast extract | 96 g | 480 g |
| glycerol 100% | 16 ml | 80 ml |
Continue with Protocol 2, preparation of whole cellular and soluble protein extracts.
© 2003 by V. Sievert, Konrad Büssow last changed 12 Sep 2006