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)

2. fill up to 1 L with deionized water
3. mix well by shaking
4. 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
5. for plates, wait until you can touch the bottle (<60 °C, clean bench!)
6. add antibiotics (1 µL/mL) and shake (gloves!)

TB medium

1. components 1:

   4 mL/L glycerol

   12 g/L tryptone

   24 g/L yeast extract

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

   0.17 M KH₂PO₄

   0.72 M K₂HPO₄

6. dissolve in 100 mL deionized water and sterilize it by passing it through a filter
7. after autoclaving and cooling down, add sterile phosphate solutions

Hartmans minimal medium (HM)

SOC

1. components

   0,5 % yeast extract

   2 % tryptone

   10 mM NaCl

   2.5 mM KCl

   20 mM MgSO₄

2. fill up with deionized water
3. adjust to pH 7.5 with NaOH
4. after autoclaving, add 20 mM sterile glucose solution (filter sterilization)

Agar Chips

Cell preparation

1. over night culture of sensor cells (50 mL in a 250 mL flask with) max. 16 h
2. centrifuge all 50 mL by 3000 g for 10 min.
3. discard the supernatant
4. re-suspend the pellet in 1 mL medium at 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 medium with the cells by inverting gently
2. pour it in the chip form, avoiding bubble formation (!)
3. wait for ca 20 min until the agar has solidified
4. cut out the chips with a scalpel
5. put ever two chips into a labeled petri dish
6. incubate for 1 h at 37 °C

Transformation

Heat Shock

1. thaw cells on ice
2. add 1 µL of plasmid DNA
3. incubate on ice for 30 min
4. heat shock at 42 °C for 60 s
5. incubate on ice for 5 min
6. add 200 µL of SOC media
7. incubate at 37 °C for 2 h
8. plate 20  and 200 µL on plates supplemented 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 a small plastic pipette to place the cells
5. pulse: 2.5 kV, 200-400 Ω, 25 μF (for E.coli)
6. immediatly add 1 mL LB and incubate for 2 h at 37 °C
7. plate 50 μL on selective medium plate
8. 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

→ 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