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)
M9 minimal medium (M9)
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 at RT (21 °C).
- discard the supernatant
- re-suspend the pellet in 1 mL tempered (~21 °C) LB-medium .
Agar preparation
- autoclave 50 mL medium with 1.5 % (w/v) agarose (has to be multiplied with the number of chips prepared).
- 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 approximately 20 min until the agar has solidified.
- cut out the chips with a scalpel.
- put two chips into a labeled petri dish and store additional 4 chips in labeled petri dishs in the refrigerator.
- incubate two chips for 1 h at 37 °C prior to induction.
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
The Gibson Assembly was conducted according to the protocol published by New England Biolabs.
- Set up the reaction according to the table below on ice (2-3 fragment assembly).
- Incubate samples in a thermocycler at 50°C for 15 minutes when 2 or 3 fragments are being assembled or 60 minutes when 4-6 fragments are being assembled. Following incubation, store samples on ice or at –20°C for subsequent transformation.
- Transform NEB 5-alpha Competent E. coli cells with 2 μl of the assembly reaction, following the transformation protocol.
Total Amount of Fragments | 0.02-0.5 pmols
|
Gibson Assembly Master Mix (2X) | 10 µl
|
Deionized H2O | 10-X µl
|
Total Volume | 20 µl
|
SDS-PAGE
Cell preparation
- lysis cell pellet in lysis buffer
- centrifuge for 15 min at 13.000 rpm
- mix the supernatant with 2x lammli buffer with β-mercaptoethanol
- denatured for 5 min at 95 °C
- sample to the gel
For some SDS-PAGEs, we used BioRad ready made gels.
The recipe of the self-made SDS is as follows:
1.5x Buffer
- 1.5 M Tris-Cl pH = 8.8
- in 1 L is 40 ml 10 % SDS
Gels
| 0.75 mm 12 % RUNNING Gel
| 1 mm 4 % STACKING Gel
|
| 1x | 2x | 4x
| 1x | 2x | 4x
|
H2O
| 1.65 mL | 3.3 mL | 6.6 mL
| 1.5 mL | 3 mL | 6 mL
|
1.5x Gel Buffer
| 1.3 mL | 2.6 mL | 5.2 mL
| 0.65 mL | 1.3 mL | 2.6 mL
|
30 % Acrylamide (37.5:1)
| 2 mL | 4 mL | 8 mL
| 0.325 mL | 0.65 mL | 1.3 mL
|
10 % APS
| 50 µL | 100 µL | 200 µL
| 25 µL | 50 µL | 100 µL
|
TEMED
| 10 µL | 20 µL | 40 µL
| 5 µL | 10 µL | 20 µL
|
Run Gel
run the gel for 10 min at 60 V and after that for ca. 60 min at 120 V
|