Team:BOKU-Vienna/Protocol
Protocol
Materials
LB medium
| Name | Amount |
|---|---|
| Peptone | 10 g |
| Yeast Extract | 5 g |
| NaCl | 5 g |
| RO-water | 1000 mL |
LB agar
| Name | Amount |
|---|---|
| Peptone | 10 g |
| Yeast Extract | 5 g |
| NaCl | 5 g |
| RO water | 986 mL |
| Agar | 14 g |
Methods
Preparation of chemical competent E. coli
• E. coli strain DH10B is inoculated from a glycerol stock onto an LB plate; the inoculum is streaked on the plate using a loop so as to obtain individual colonies. The plate is incubated overnight at 37 °C.
• Inoculate 10 mL of LB medium with a single colony and incubate the flask overnight in a shaker-incubator (37°C, shaking 180 rpm).
• The following day, transfer 2 mL of this culture to a flask containing 200 mL LB medium and incubate for about 2h until OD600 raches 0.6.
• Cool down the cells on ice for 10 minutes. The cells are pelleted in a centrifuge for 5 min at 4,500 rpm (4,000 x g) at 4°C. The cells are resuspended in 0.4 volume of ice-cold TFB1*.
• Repeat the centrifugationstep. Resuspend the pellet in 1/25 volume of ice-cold TFB2**.
• the cells are aliquoted 100 µL per tube and stored at -80 °C.
*
