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From 2014.igem.org

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Revision as of 21:15, 17 October 2014

6^th June

• We continue our experiments to have BL21 without plasmid.


• Preparation of LB + Amp and LB + Chlo plates.

Protocol :

• ELP VPGXG-20 and VPGXG-40


1. Take a single colony and inoculate 250 mL LB + Amp at 37°C overnight
2. Next morning, inoculate 1L LB + Yeast Extract (5 g/L) + Glycerol (1 g/L) + Amp (100 mg/L) in order to have A600: 0.2. We can use Terrific Broth instead of LB.
3. Grow cells until A600 : 0.7-0.9
4. Take out 10 mL of culture, centrifuge 5min at 14,000 rpm and 4°C, discard supernatant and store pellet at -20°C (this is the uninduced time point).
5. Add 1mM (final concentration) of IPTG
6. After adding 1mM IPTG, re-incubate remaining cultures at 25°C shaking with 180rpm during 21h.
7. Collect 1ml fraction for the A600 (at t=1h, t=2h, t=3h, t=4h, t=5h and t=21h after induction)


• ELP VPGXG-60
1. Take a single colony and inoculate 250 mL LB + Amp at 37°C overnight
2. Next morning, inoculate 1L LB + Yeast Extract (5 g/L) + Glucose (1 g/L) + Amp (100 mg/L) in order to have A600: 0.2. We can use Terrific Broth instead of LB.
3. Grow cells until A600 : 0.7-0.9
4. Take out 10 mL of culture, centrifuge 5min at 14,000 rpm and 4°C, discard supernatant and store pellet at -20°C (this is the uninduced time point).
5. Add 1mM (final concentration) of IPTG
6. After adding 1mM IPTG, re-incubate remaining cultures at 25°C shaking with 180rpm during 21h.
7. Collect 1ml fraction for the A600 (at t=1h, t=2h, t=3h, t=4h, t=5h and t=21h after induction)


• We do a 30mL preculture of LB + yeast extract 5g/L + 1 g/L glycerol + Amp of (VPGMG-20) and (VPGMG-40) at 37°C overnight.