materials, equipment andreagents
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Operation Step
2) to the calibration interface, cursor to DO, "span" file, set the reading to 100, adjust several times, until stable to 100, go to the menu interface.
5． Other parameter settings.
1) to adjust the cursor to Agit gear, setting a minimum of 200r/min. Adjust the cursor to DO gear, set the parameters to 10/20/30, adjust to the oxygen-dissolved joint detection. Adjust the cursor to Agit gear and set the maximum value to 800r/min.
2) to adjust the cursor to pH gear, set the parameter 7.0, adjust to automatic detection.
3) to the acid (alkali) bottle and turn on the automatic detection system.
6． Inoculation.
1) wraps a cotton ball filled with ethanol around the inoculation jacket and loosens the lid of the inoculated mouth before igniting the flame.
2) ignites the cotton ball, removes the lid, immediately adds 1mg/mL canamycin (Km) storage fluid to the fermentation tank in the flame, so that the final concentration of 100 mg/mL, as well as the various partsmedium, record pH display value, sampling, measurement actual value, pH Setpoint display value, vaccination.
3) soak the lid in ethanol, after inoculation, use the camera to put the lid exactly back in place, tighten.
7． Open the dedicated software that records the experimental process.
8． Culture to OD600;10.00 (around 6-8hrs), add IPTG to the final concentration of 1mM, continue fermentation culture, glucose concentration dropped to 5g/L when added glucose. If necessary, the growth status of bacteria and contamination can be checked at any time.
9． Fermentation ends when the pH rises to 7.3 (24hrs).
10． Bacteria collection: after fermentation, 4 degrees C 8000r/min centrifugation 10min, collection of bacteria, -20 degrees C to save standby.
11． During fermentation, every two hours to record dissolved oxygen, speed (record average), determination of OD600, sample 5 ml into the weight of the centrifuge tube (3 each), before and after the filling to sample.
(Responsible Editor: King Han Kunlun)
