Team:METU Turkey/Results Discussion/Protein expression

From 2010.igem.org

Latest revision as of 03:42, 28 October 2010

Main Steps/Time Table

- Pre-culturing/5 hours
- Culturing/5 hours
- IPTG induction/15 hours

Materials

- LB medium with a suitable antibiotic
- TB medium with a suitable antibiotic
- IPTG

- 2-liter flask
- Incubator with shaker
- Centrifuge

Check List Procedure

- Culture transformed E.coli BL21 for 5 hrs at 37 C in 10 LB medium containing antibiotic
- Inoculate 4 ml pre-cultured cells into 400 ml of TB medium containing antibiotic in a 2-liter cultivation flask
- Culture it for 5 hrs at 37 C with a rotary shaker at 180 rpm!
- Add 0.5 mM IPTG in a final concentration
- Continue the cultivation for 15 hrs at 22 C.
- Harvest the cells with centrifuge.

Alternative Check List Procedure

- Inoculate single colony for 5 hrs at 37 C in 10 mL LB medium containing antibiotic
- Inoculate 4 ml pre-cultured cells into 400 ml of TB medium containing antibiotic in a 2-liter cultivation flask
- Culture it for 5 hrs at 37 C (OD600 to 0.5 - 0.6) with a rotary shaker at 180 rpm
- Add 1 mM IPTG in a final concentration ( 0.5- 2.0 mM recomended for pT7)
- Continue the cultivation for 3-4 h at 37 C (15 hrs at 22 C.)
- Harvest the cells with centrifuge.

Solutions

- TB (Terrific Broth) see Procedure [Media]

Notes

Incubation temperature incubation time
15°C overnight
20°C overnight
25°C overnight
30°C 5-6 h
37°C 3-4 h

reference: http://www.embl.de/pepcore/pepcore_services/protein_expression/ecoli/

Cell Lysis

Materials:

- water bath
- vortex
- pipet 10 ul
- ice

Check List Procedure

- For 1 ml cell culture;

- resuspend the cell pellets in 1 ml 2-5% SDS
- vortex 1 minute
- incubate in 50 c water bath for 15 min
- put in ice for 1 min
- add 10 ul from each of
- 1 mg/ml RNAse A stock (final concentration: 10 ug/ml)
- 0.5 mg/ml DNase I stock (final concentration: 5 ug/ml)
- 100 mM MgCI2 stock (final concentration: 1mM)
- incubate on ice for 15 min

Solutions

- 1 mg/ml RNAse A stock
- 0.5 mg/ml DNase I stock
- 100 mM MgCI2 stock
- 2-5% SDS