Team:Wageningen UR/Protocol/StartupHepB
From 2012.igem.org
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== Reagents & Materials == | == Reagents & Materials == | ||
- | For a | + | For a 50 mL culture: |
reagents: | reagents: | ||
<ul> | <ul> | ||
- | <li> | + | <li>60 mL of LB</li> |
<li>kanamycin</li> | <li>kanamycin</li> | ||
- | <li>0. | + | <li>0.25 mL of IPTG stock solution 250 mM</li> |
<li>MQ-water</li> | <li>MQ-water</li> | ||
</ul> | </ul> | ||
Line 17: | Line 17: | ||
<ul> | <ul> | ||
<li>Pipettes and pipettes points</li> | <li>Pipettes and pipettes points</li> | ||
- | <li> | + | <li>250 ml Erlenmeyer</li> |
<li>Greiner tubes</li> | <li>Greiner tubes</li> | ||
<li>Freezer</li> | <li>Freezer</li> |
Revision as of 11:24, 13 September 2012
Reagents & Materials
For a 50 mL culture:
reagents:
- 60 mL of LB
- kanamycin
- 0.25 mL of IPTG stock solution 250 mM
- MQ-water
materials:
- Pipettes and pipettes points
- 250 ml Erlenmeyer
- Greiner tubes
- Freezer
- Centrifuge for Greiner tubes
Procedure
- Prepare a Erlenmeyer flask with 50mL of LB-medium and keep it at 37°C
- Pick E.coli BL21 from a plate and grow them over night in a 10 mL LB culture containing 50mg/L Kanamycin at 37°C
- Inoculate the large flask with 1 mL of the overnight culture and grow at 37°C for 2.5h until the OD600 reaches approx. 0.6
- Induce with 1.25 mM IPTG (0.25 mL of a 250 mM stock)
- Incubate at 37°C for 4 h
- Centrifuge the cells in 50ml greiner tubes at 4700 rpm for 18 minutes
- Decant the supernatant and resuspend the pellets in ± 10 mL washing buffer each
- Centrifuge again at 4700 rpm for 18 minutes
- Decant the supernatant and centrifuge for another minute
- Pipette any liquid to clear the tube of supernatant
- (possible to freeze the cells to -20°C at this point)