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From 2012.igem.org

Growing Culture

Reagents & Materials

For a 100 mL batch:

Reagents:

• 110 mL of LB
• kanamycin
• 0.5 mL of IPTG stock solution 250 mM
• MQ-water

Materials:

• Pipettes and pipettes points
• 500 ml Erlenmeyer
• Greiner tubes
• Freezer
• Centrifuge for Greiner tubes

Procedure

1. Prepare a Erlenmeyer flask with 0.1 L of LB-medium and keep it at 37°C
2. 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
3. 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
4. Induce with 1.25 mM IPTG (0.5 mL of a 250 mM stock)
5. Incubate at 37°C for 4 h
6. Centrifuge the cells in 50ml greiner tubes at 4700 rpm for 18 minutes
7. Decant the supernatant and resuspend the pellets in ± 20 mL mQ water each
8. Centrifuge again at 4700 rpm for 18 minutes
9. Decant the supernatant and centrifuge for another minute
10. Pipette any liquid to clear the tube of supernatant
11. (possible to freeze the cells to -20°C at this point)

Next: CCMV dialysis