Media
LB medium
- weight components
- 5 g/L NaCl
- 10 g/L tryptone
- 5 g/L yeast extract
- (15 g/L agar for plates)
- fill up to 1 L with deionized water
- mix well by shaking
- autoclave
- autoclaving tape, caps slightly unscrewed
- base of the pot has to be covered with deionized water
- close lid
- heat level 3 until the pressure valve opens
- reduce heat level to 1.5
- set timer to 20 minutes
- turn heater off
- wait until the pressure valve retracts (30-45 minutes)
- open, close caps & shake
- for plates, wait until you can touch the bottle (<60 °C, clean bench!)
- add antibiotics and shake (gloves!)
Hartmans minimal medium (HM)
Agar Chips
Cell preparation
- over night culture of sensor cells (50 ml in a 250 ml flask with) max. 16h
- centrifuge all 50 ml by 3000 xg for 10 min.
- discard the supernatant
- resuspend the pellet in 1 ml warm medium (RT)
Agar preparation
- autoclave 50 ml medium with 1,5 % agarose
- cool it down to 45 °C in a water bath
Chip production
- mix the cooled dowm medium with the cells by inverting gently
- pour it in the chip form without bubbles
- wait for ca 20 min till it is solid
- cut out the chips with a scalpel
- put ever two chips into a labeled petri dish
Transformation
Heat Shock
- thaw cells on ice
- add 1 µl of plasmid DNA
- incubate on ice for 30'
- heat shock at 42 °C for 60
- incubate on ice for 5'
- add 200 µl of SOC media
- incubate at 37 °C for 2 h
- plate 20 and 200 µl on plates with the appropiate antibiotic
Electroporation
PCR
Colony PCR
QuikChange
Clonings
Restriction Digest
Ligation
Gibson Assembly
SDS-PAGE
For some SDS-PAGEs we used BioRad ready made gels.
The recipe of the self-made SDS is as follows:
3.5x Buffer
Gels
| 1mm 12 % RUNNING Gel
| 1mm 8 % RUNNING Gel
| 1mm 4 % STACKING Gel
|
| 1x | 2x | 4x
| 1x | 2x | 4x
| 1x | 2x | 4x
|
H2O
| 1.669 ml | 3.337 ml | 6.674 ml
| 2.45 ml | 4.9 ml | 9.8 ml
| 1.421 ml | 2.842 ml | 5.684 ml
|
3.5x Gel Buffer
| 1.613 ml | 3.225 ml | 6.45 ml
| 1.613 ml | 3.225 ml | 6.45 ml
| 0.7 ml | 1.4 ml | 2.8 ml
|
30 % Acrylamide (37.5:1)
| 2.344 ml | 4.687 ml | 9.374 ml
| 1.563 ml | 3.125 ml | 6.25 ml
| 0.329 ml | 0.658 ml | 1.316 ml
|
10 % APS
| 22.5 µl | 45 µl | 90 µl
| 22.5 µl | 45 µl | 90 µl
| 10.5 µl | 21 µl | 42 µl
|
TEMED
| 2.25 µl | 4.5 µl | 9 µl
| 2.25 µl | 4.5 µl | 9 µl
| 4.9 µl | 9.8 µl | 19.6 µl
|
|