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 (1 µL/mL) and shake (gloves!)
TB medium
- Components 1:
- 4 mL/L glycerol
- 12 g/L tryptone
- 24 g/L yeast extract
- Fill up to 900 mL with deionized water
- Mix well by shaking
- Autoclave
- Components 2:
- 0.17 M KH2PO4
- 0.72 M K2HPO4
- Dissolve in 100 mL deionized water and sterilize it by passing it through a filter
- After autoclaving and cooling down, add sterile phosphate solutions
Hartmans minimal medium (HM)
SOC
- Components
- 0,5 % yeast extract
- 2 % tryptone
- 10 mM NaCl
- 2.5 mM KCl
- 20 mM MgSO4
- Fill up with deionized water
- Adjust to pH 7.5 with NaOH
- After autoclaving, add 20 mM sterile glucose solution (filter sterilization)
Agar Chips
Cell preparation
- Over night culture of sensor cells (50 mL in a 250 mL flask with) max. 16 h
- Centrifuge all 50 mL by 3000 g for 10 min.
- Discard the supernatant
- Re-suspend the pellet in 1 mL medium at 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 medium with the cells by inverting gently
- Pour it in the chip form, avoiding bubble formation (!)
- Wait for ca 20 min until the agar has solidified
- Cut out the chips with a scalpel
- Put ever two chips into a labeled petri dish
- Incubate for 1 h at 37 °C
Transformation
Heat Shock
- Thaw cells on ice
- Add 1 µL of plasmid DNA
- Incubate on ice for 30 min
- Heat shock at 42 °C for 60 s
- Incubate on ice for 5 min
- Add 200 µL of SOC media
- Incubate at 37 °C for 2 h
- Plate 20 and 200 µL on plates supplemented with the appropiate antibiotic
Electroporation
- Add 1 μL plasmid to electrocompetent cells
- Put DNA/ cell suspension in electroporation cuvette
- Wipe dry the electroporator
- Use a small plastic pipette to place the cells
- Pulse: 2.5 kV, 200-400 Ω, 25 μF (for E.coli)
- Immediatly add 1 mL LB and incubate for 2 h at 37 °C
- Plate 50 μL on selective medium plate
- Centrifuge the rest (3000 g, 20 min), discard supernatant, re-suspend the pellet in 50 μL LB and plate it on selective medium plate
PCR
Colony PCR
with GoTaq Mast Mix
- 12.5 µl GoTaq Master Mix
- 1 µl primer_F
- 1 µl primer_R
- pick colony with tip and suspend in PCR tube
- 9.5 µl ddH2O
parameter | duration | temp [°C]
|
denature | 5:00 | 95
|
anneal | 00:30 | 56
|
elongate | 01:00 per kb | 72
|
denature | 00:30 | 95
|
elongate | 05:00 | 72
|
store | forever | 8
|
→ 30 cycles
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
| 1 mm 12 % RUNNING Gel
| 1 mm 8 % RUNNING Gel
| 1 mm 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
|
|