Revision as of 10:20, 10 September 2012

Wageningen UR

Growing Culture

Prepare a Erlenmeyer flask with 0.1 L 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 (0.5 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 ± 20 mL mQ water 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)

@@ Line 13: / Line 13: @@
 <li>Induce with '''1.25 mM IPTG''' (0.5 mL of a 250 mM stock)</li>
 <li>Incubate at 37°C for 4 h</li>
-<li>Centrifuge the cells in two 500 mL flask in the Sorvall centrifuge at 5000 rpm for 15 minutes</li>
+<li>Centrifuge the cells in 50ml greiner tubes  at 4700 rpm for 18 minutes</li>
 <li>Decant the supernatant and resuspend the pellets in ± 20 mL mQ water each</li>
-<li>Combine the resuspended cells in a 50 mL Greiner  tube and centrifuge at 5000 rpm for 15 minutes</li>
+<li>Centrifuge again at 4700 rpm for 18 minutes</li>
 <li>Decant the supernatant and centrifuge for another minute</li>
 <li>Pipette any liquid to clear the tube of supernatant</li>