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Team:METU Turkey/Results Discussion/Protein expression
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Serapmemik (Talk | contribs) (New page: <br>MAIN STEPS/TIME TABLE <br>- Pre-culturing/5 hours <br>- Culturing/5 hours <br>- IPTG induction/15 hours <br>Materials <br>- LB medium with a suitable antibiotic <br>- TB medium with ...) |
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| - | <br>MAIN STEPS/TIME TABLE | + | '''<br>MAIN STEPS/TIME TABLE''' |
<br>- Pre-culturing/5 hours | <br>- Pre-culturing/5 hours | ||
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<br>- Centrifuge | <br>- Centrifuge | ||
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<br>- Culture transformed E.coli BL21 for 5 hrs at 37 C in 10 LB medium containing antibiotic | <br>- Culture transformed E.coli BL21 for 5 hrs at 37 C in 10 LB medium containing antibiotic | ||
<br>- Inoculate 4 ml pre-cultured cells into 400 ml of TB medium containing antibiotic in a 2-liter cultivation flask | <br>- Inoculate 4 ml pre-cultured cells into 400 ml of TB medium containing antibiotic in a 2-liter cultivation flask | ||
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<br>- Harvest the cells with centrifuge. | <br>- Harvest the cells with centrifuge. | ||
| - | <br>Alternative Check List Procedure | + | '''<br>Alternative Check List Procedure''' |
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<br>- Inoculate single colony for 5 hrs at 37 C in 10 mL LB medium containing antibiotic | <br>- Inoculate single colony for 5 hrs at 37 C in 10 mL LB medium containing antibiotic | ||
<br>- Inoculate 4 ml pre-cultured cells into 400 ml of TB medium containing antibiotic in a 2-liter cultivation flask | <br>- Inoculate 4 ml pre-cultured cells into 400 ml of TB medium containing antibiotic in a 2-liter cultivation flask | ||
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<br>- Harvest the cells with centrifuge. | <br>- Harvest the cells with centrifuge. | ||
| - | <br>Solutions | + | '''<br>Solutions''' |
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<br>- TB (Terrific Broth) see Procedure [Media] | <br>- TB (Terrific Broth) see Procedure [Media] | ||
| - | <br>Notes | + | '''<br>Notes''' |
<br>Incubation temperature incubation time | <br>Incubation temperature incubation time | ||
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<br>- ice | <br>- ice | ||
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<br>- For 1 ml cell culture; | <br>- For 1 ml cell culture; | ||
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<br>- incubate on ice for 15 min | <br>- incubate on ice for 15 min | ||
| - | <br>Solutions | + | '''<br>Solutions''' |
<br>- 1 mg/ml RNAse A stock | <br>- 1 mg/ml RNAse A stock | ||
Revision as of 03:38, 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
