Team:METU Turkey/Results Discussion/Protein expression

From 2010.igem.org

(Difference between revisions)
(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>- IPTG induction/15 hours
<br>- IPTG induction/15 hours
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<br>Materials
+
'''<br>Materials'''
 +
 
<br>- LB medium with a suitable antibiotic
<br>- LB medium with a suitable antibiotic
<br>- TB medium with a suitable antibiotic
<br>- TB medium with a suitable antibiotic
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<br>- Centrifuge
<br>- Centrifuge
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<br>Check List Procedure
+
'''<br>Check List Procedure'''
 +
 
<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.
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<br>Alternative Check List Procedure
+
'''<br>Alternative Check List Procedure'''
 +
 
<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.
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<br>Solutions
+
'''<br>Solutions'''
 +
 
<br>- TB (Terrific Broth) see Procedure [Media]
<br>- TB (Terrific Broth) see Procedure [Media]
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<br>Notes
+
'''<br>Notes'''
<br>Incubation temperature incubation time  
<br>Incubation temperature incubation time  
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<br>Cell Lysis  
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'''<br>Cell Lysis'''
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<br>Materials:
+
'''<br>Materials:'''
<br>- water bath
<br>- water bath
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<br>- ice
<br>- ice
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<br>Check List Procedure
+
'''<br>Check List Procedure'''
<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
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<br>Solutions
+
'''<br>Solutions'''
<br>- 1 mg/ml RNAse A stock
<br>- 1 mg/ml RNAse A stock

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

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