Team:Groningen/Protocols and Protocols/IPTGinduction
Blue Bio Energy
IPTGinduction
Induction of IPTG promotor to overexpress the protein coupled to the IPTG promotor
ML-I
Preculture
Inoculate 2-3 mL LB + antibiotic on 37°C 200 rpm overnight.
Preparation SDS-PAGE gel
Make SDS-PAGE gel with according separation and loading gels:
Separation gel (10%, 5mL)
Quantity (mL)
Acrylamide 40%
1.25
Tris-Hcl 1,5M pH=8,8
1.25
H2O
2.42
SDS 10%
0.05
TEMED
0.01
APS 10%
0.02
Separation gel
Loading gel (2,5 mL)
Quantity (mL)
Acrylamide 40%
0.25
Tris-Hcl 1,5M pH=8,8
0.625
H2O
1.585
SDS 10%
0.025
TEMED
0.005
APS 10%
0.01
Loading gel
Culture
Inoculate 20mL LB + antibiotic with 100 times diluted overnight culture from your preculture step.
When the culture has an OD of 0.5-0.6 add 1mM of IPTG.
Before adding IPTG take control sample of 1mL and measure its OD.
Take 1mL sample 1 hours after IPTG induction and measure its OD.
Take 1mL sample 2 hours after IPTG induction and measure its OD.
Take 1mL sample 3 hours IPTG induction and measure its OD.
Take 1mL sample 12 hours IPTG induction and measure its OD.
Pellet the cells of each sample by centrifuging at 11000rpm for 1 minute.
Remove supernatant and resuspend in 300 µL TE bufffer (10mM Tris pH8.0 ; 1mM EDTA).
Sonicate 4x 10 seconds pulse; 5 seconds pause; amplitude of 35% (keep your tube in ice during sonification).
Centrifuge 10 min at 14000rpm at 4°C.
Place supernatant in a new tube and resuspend pellet with 300 µL TE buffer
.
Add 7µL 4X loading buffer to 21µL of each sample.
Denaturate each sample 5 min at 95°C.
Load 15µL on SDS-PAGE gel.
Run gel first at 110V until sample reaches separation gel, afterwards on 200V.
Stain gel with Coomassie (40% MeOH, 10% Acetic Acid, 0,1% Blue R250) for 20 minutes.
Destain (10% Acetic Acid, 20% EtOH) 2x 10 minutes, afterwards destain until only the bands are visible on the gel.