Team:Aachen/Notebook/Protocols

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Contents


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)

M9 minimal medium (M9)

Aachen_Components_M9_Minimal_Medium.png

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 at RT (21 °C).
  3. Discard the supernatant
  4. Re-suspend the pellet in 1 mL tempered (~21 °C) LB-medium .


Agar preparation

  1. Autoclave 50 mL medium with 1.5 % (w/v) agarose (has to be multiplied with the number of chips prepared).
  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 approximately 20 min until the agar has solidified.
  4. Cut out the chips with a scalpel.
  5. Put two chips into a labeled petri dish and store additional 4 chips in labeled petri dishs in the refrigerator.
  6. Incubate two chips for 1 h at 37 °C prior to induction.

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
parameter duration temp [°C]
denature5:0095
anneal00:3056
elongate01:00 per kb72
denature00:3095
elongate05:0072
storeforever8

→ 30 cycles

QuikChange

Clonings

Restriction Digest

Ligation

Gibson Assembly

The Gibson Assembly was conducted according to the protocol published by New England Biolabs.

  1. one Set up following reaction on ice (2-3 fragment assembly):
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
  1. two 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.
  1. three Transform NEB 5-alpha Competent E. coli cells (provided with the kit) with 2 μl of the assembly reaction, following the transformation protocol.

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