Media

LB medium

1. weight components

• 5 g/L NaCl
• 10 g/L tryptone
• 5 g/L yeast extract
• (15 g/L agar for plates)

1. fill up to 1 L with deionized water
2. mix well by shaking
3. autoclave
   1. autoclaving tape, caps slightly unscrewed
   2. base of the pot has to be covered with deionized water
   3. close lid
   4. heat level 3 until the pressure valve opens
   5. reduce heat level to 1.5
   6. set timer to 20 minutes
   7. turn heater off
   8. wait until the pressure valve retracts (30-45 minutes)
   9. open, close caps & shake
4. for plates, wait until you can touch the bottle (<60 °C, clean bench!)
5. add antibiotics and shake (gloves!)

TB medium

1. components 1:

• 4 ml/L glycerol
• 12 g/L tryptone
• 24 g/L yeast extract

1. fill up to 900 ml with deionized water
2. mix well by shaking
3. autoclave
4. components 2:

• 0.17 M KH_2PO_4
• 0.72 M K_2HPO_4

1. dissolve in 100 ml deionized water and sterile filter it
2. after autoklave and cooling down add sterile filtered phosphates

Hartmans minimal medium (HM)

SOC

1. components

• 0,5 % yeast extract
• 2 % tryptone
• 10 mM NaCl
• 2,5 mM KCl
• 20 mM MgSO₄

1. fill up with deionized water
2. adjust to pH 7,5 with NaOH
3. after autoklave add 20 mM sterile filtered glucose

Agar Chips

Cell preparation

1. over night culture of sensor cells (50 ml in a 250 ml flask with) max. 16h
2. centrifuge all 50 ml by 3000 xg for 10 min.
3. discard the supernatant
4. resuspend the pellet in 1 ml warm medium (RT)

Agar preparation

1. autoclave 50 ml medium with 1,5 % agarose
2. cool it down to 45 °C in a water bath

Chip production

1. mix the cooled dowm medium with the cells by inverting gently
2. pour it in the chip form without bubbles
3. wait for ca 20 min till it is solid
4. cut out the chips with a scalpel
5. put ever two chips into a labeled petri dish
6. incubate for 1h by 37°C

Transformation

Heat Shock

1. thaw cells on ice
2. add 1 µl of plasmid DNA
3. incubate on ice for 30'
4. heat shock at 42 °C for 60
5. incubate on ice for 5'
6. add 200 µl of SOC media
7. incubate at 37 °C for 2 h
8. plate 20  and 200 µl on plates with the appropiate antibiotic

Electroporation

1. add 1 μl plasmid to electrocompetent cells
2. put DNA/ cell suspension in electroporation cuvette
3. wipe dry the electroporator
4. use small plastic pipette to place the cells
5. puls: 2,5 kV, 200-400Ω, 25 μF (for E.coli)
6. immediatly add 1 ml LB and incubate for 2h by 37 °C
7. plate 50 μl on selectiv medium plate
8. centrifuge the rest (3000 xg, 20 min), discard supernatant, resuspend the pellet in 50 μl and plate it on selectiv medium plate

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