Add 0.1 times the volume of water to rehydrate the dried DNA from twist. The resuspended DNA was aliquoted into ten equal portions, with one aliquot stored at -20°C for convenient access, while the remaining aliquots were frozen at -80°C for long-term preservation.
Pick E.coli BL21 DE3 out of the -80°C freezer and culture on LB agar plates. And place the plate into the incubator.
Colony picking of the colonies that have grown on the agar
plates. These colonies will be used for competent cells preparation. Transformation of plasmid in competent
cells.
1. Cultivate a small quantity of bacteria: Inoculate two colonies from each plate
into culture tubes containing 4 ml of LB medium and place them in a shaking incubator for 12-16 hours (with
antibiotics in the
medium).
2. Scale up the culture: Transfer 3 ml of the bacterial culture from the culture tubes
to conical flasks ontaining 200 ml, and incubate in a shaking incubator for 2-3 hours, until the OD value
reaches between
0.6-0.8.
3. Separate bacteria from the culture by high-speed centrifugation.
4. Disrupt bacteria and separate bacterial remains and proteins by high-speed
centrifugation.
5. Purify the protein using His-tag purification.
Fig. 1 Before improvement
Fig. 2 After improvement