Team:Freiburg/Protocols/Competent Cells
competent cells (new)
e.g. protocol for DNA-isolation
remarks concerning the protocol (author, adapted from etc.)
material: chemicals, used kits, …
duration: … min
Protocol
- streak out E. coli cell on LB plate without (or with appropriate antibiotics), grow over night at 37°C
- inocculate 5 ml o/n culture with one colony
- inocculate 100 ml LB with 1 ml o/n culture, grow for 2-3 h until an OD(600)=0,4 to 0,6
- transfer bacteria into pre-cooled and sterile centrifuge tube (250 ml) and pellet bacteria 5 min (5000 rpm; 4°C)
- discard supernatant
from now on: work in cold room
- resuspend pellet in 20 ml cold TFB1 with cut tips
- incubate 5 min on ice
- centrifuge 5 min (5000 rpm; 4°C)
- resuspend pellet in 4 ml cold TFB2 with cut tips
- aliquot 50 ml in screw cap eppis and freeze in liquid nitrogen
- store at -80°C
buffer preparation
| TFB1 | 200ml |
|---|---|
| 30 mM KAc | 0,589 g |
| 50 mM MnCl2 | 1,6919 g |
| 100 mM KCl | 1,482 g |
| 10 mM CaCl2 | 0,294 g |
| 15 % Glycerol | 30 g |
* sterile filtrate and store at 4°C
| TFB2 | 200 ml |
|---|---|
| 10 mM MOPS | 0,419 g |
| 10 mM KCl | 0,149 g |
| 75 mM CaCl2 | 2,205 g |
| 15 % Glycerol | 30 g |
* autoclave and store at 4°C
