Bacteria Culture Protocol

Bacteria Culture Protocol
By 徐晓政

1、TBS Medium Preparation:
Prepare 1L of TBS medium contains:
Tryptone 12g
Yeast extract 24g
NaCl 5g
Sodium Succinate 5g
Glycerol 20ml
Adjust PH to 7.0 after totally solved.
Sterilize with 121OC, 15min. When medium Tm is below 60OC, add 1/1000 Glucose&MgSO4 store
solution ( Dissolve 25g Glucose and 12.3g MgSO4·7H2O in ddwater to a final volume of 50ml,
then sterile by filtered with a 0.22um membrane ).
2、TBS Medium Culture Protocol (for DH5a)
Inoculate a single clone to 50ml TBS medium in a 250ml baffled flask . Incubate in 250rpm/37 OC
for 24h, Be sure that air inner the flask can exchange well with outside.
3、Auto‐induction Medium (AIM) Preparation (for BL21de3)
Prepare 1L of AIM contains:
Tryptone 12g
Yeast extract 24g
Sodium Succinate 4g
Sodium Citrate 6g
Glycerol 20ml
Lactose 5g
Adjust PH to 7.0 after totally solved.
Sterilize with 121OC, 15min. When medium Tm is below 60OC, add 1/1000 Glucose&MgSO4 store
solution ( Dissolve 25g Glucose and 12.3g MgSO4·7H2O in ddwater to a final volume of 50ml,
then sterilize by filtered with a 0.22um membrane ).
4、Auto‐induction Medium (AIM) Expression Method
Inoculate a single clone to 50ml TBS medium in a 250ml baffled flask. Incubate in 250rpm/37 OC
for 6h, then change Tm to your induction Tm and continue culturing until OD600 reaches 20‐40
for full induction. Be sure that air inner the flask can exchange well with outside.
Note: Collect bacterium in a slightly lower centrifuge speed to prevent from contaminated by
medium ingredients or metabolites, we recommend 3000rpm for J6‐MI (