Team:Tuebingen/Methods

Methods

Chemically Competent E. coli Cells

  • autoclave 250mL LB in Erlenmeyer beaker
  • plate cells from cell stock on agarose plate with appropriate antibiotics
  • inoculate one clone in 5mL of LB with antibiotics, grow at 37°C o/n
  • expand to 500mL and grow until OD600nm = 0.35
  • transfer into 50mL Falcon tubes
  • refrigerate centrifuge at 4°C, chill 2x 25mL 0.1M CaCl2 (Falcon tubes) and 5mL 0.1M CaCl2 with 10% Glycerol
  • spin down cells at 2,000g for 10Min at 4°C
  • resuspend 5 Falcon cell pellets in 25mL ice cold 0.1M CaCl2
  • spin down at 2,000g for 10Min at 4°C
  • resuspend pellet in 2x5mL (=10mL) ice cold 0.1M CaCl2 with 10% Glycerol
  • freeze 50-100μl aliquots
  • store at 20°C

DoubleDigest Restriction of gBlocks and BioBricks

  • 2μl 10x buffer (corresponding to enzymes)
  • 0,5μl restriction enzyme 1
  • 0,5μl restriction enzyme 2
  • 1μg DNA
  • fill up to 20μl with H2O

    • Method:

  • mix all components
  • incubate at 37°C for 2h
  • heatinactivate enzymes at 80°C for 10min or use for gel purification

Gelpurification

This is a modified protocol for the Wizard SV Gel and PCR CleanUp System Kit from Promega

  • Following electrophoresis, excise DNA band from gel and place gel slice in a 1,5ml microcentrifuge tube
  • Add 10μl Membrane Binding Solution per 10mg of gel slice. Vortex and incubate at 5060°C until gel slice is completely dissolved
  • Insert SV Minicolumn into Collection Tube
  • Transfer dissolved gel mixture to the Minicolumn assembly. Incubate at room temp. for 1 minute
  • Centrifuge at 16,000xg for 1 min. Discard flowthrough and reinsert Minicolumn into Collection tube
  • Add 700μl Membrane Wash Solution (ethanol added). Centrifuge at 16,000xg for 1 min
  • Discard flowthrough and reinsert Minicolumn into Collection tube
  • Repeat this step with 500μl Membrane Wash Solution. Centrifuge at 16,000xg for 5 min
  • Empty the Collection Tube and centrifuge the column assembly for 1,5 min
  • Leave the tubes open for 10 min (to let any rest of ethanol evaporate)
  • Carefully transfer the Minicolumn to a clean 1.5ml microcentrifuge tube
  • Add 30μl of NucleaseFreeWater (65°C) to the Minicolumn. Incubate at room temperature for 10 min
  • Centrifuge at 16,000xg for 1 min.
  • Incubate sample at 65°C for 5min
  • Discard Minicolumn and store DNA at 4°C or 20°C

Ligation

  • 1μl 10x ligase buffer
  • 0,5μl ligase
  • 1μl ATP
  • 0,51μl linearised vector
  • 7μl insert

    • Method:

  • Mix all components
  • Incubate overnight at 4°C
  • Gelpurify using agarose gels

Smallscale plasmid preparation

Preparation was done using the PureYield™ Plasmid Miniprep System by Promega

  • Pick colonies for overnight cultures in 23ml LB medium with antibiotic grow overnight
  • Spin down 1,5ml bacteria at maximum speed for 30 minutes, discard supernatant and add further 1.5ml, spin again and discard supernatant
  • Resuspend the bacteria pellet in 600μl H2O
  • Add 100 μl Cell Lysis Buffer, invert six times
  • Add 350 μl cold Neutralization Solution, mix thoroughly by inverting
  • Centrifuge 30s at maximum speed
  • Transfer supernatant to PureYield Minicolumn in collection tube
  • Centrifuge for 30 sec, discard the flow-through
  • Add 200 μl Endotoxin Removal Wash(ERB), centrifuge for 30sec, discard the flow-through.
  • Add 400 μl Column Wash Solution(CWC), centrifuge for 30sec, discard the flow-through.
  • Place column in eppendorf tube, add 30 μl Elution Buffer to minicolumn matrix, incubate for 1 min
  • Centrifuge column for 30s at maximum speed, flow-through contains plasmid DNA
  • Measure the DNA concentration using a Nanodrop

Testrestriction

  • 0,25μl Enzyme 1
  • 0,25μl Enzyme 2
  • 1μl 10x buffer (corresponding to enzymes)
  • 200-300ng DNA
  • Fill up with water to 10μl.

    • Method:

  • Incubate at 37°C for 1h
  • Run on 1% agarose gel

Transformation of E. coli

  • 50μl chemically competent E. coli
  • 35μl overnight ligation mix OR 1ng purified plasmid DNA

    • Method:

  • Thaw bacteria pellet on ice
  • add ligation mixture or purified plasmid to bacteria
  • incubate for 20min on ice
  • heat shock bacteria for 45s at 42°C
  • incubate bacteria on ice for 2min
  • add 700μl LB medium without antibiotics
  • incubate for 1h at 37°C
  • spin down bacteria at 2g for 2min
  • discard supernatant by tipping the tube
  • resuspend bacterial pellet in leftover supernatant (50-100μl)
  • streak bacteria onto LBagar plates with antibiotics and incubate overnight

Transformation of L. johnsonii

  • 100ml L. johnsonii (OD595=0.7)
  • 107ml Transformation buffer
  • 10μl vector DNA

    Method:

  • Resuspend pelleted bacteria (4000xg/5min) in 100ml Transformation buffer
  • Zentrifuge and resuspend the bacteria again in 5ml Transformation buffer
  • Repaet step with resuspension in 2ml Transformation buffer
  • Hold on ice for 1min
  • Electroporate 200μl bacteria in 0.2cm cuvette at 25μF and 200ohms and time constant between 4.1 and 4.5ms
  • Mix electroporated bacteria with 1ml MRS medium each
  • Incubate each bacteria solution in additional 15ml MRS medium for 18h at 37°C

Fructose test

  • Reagent preparation:
  • Deproteinizing agent 1: dissolve 1,8g of ZnSO4*7H2O ind 100 ml H2O
  • Deproteinizing Agent 2: dissolve 0,4g NaOH in 100 ml H2O
  • Color reagent: dissolve 200mg benzoic acid in 90ml H2O at 60°C.
  • dissolve 25mg Indole in it and fill up to 100ml with H2O
  • filter with 0,45μm and store at 4°C
  • Procedure:
  • Centrifuge broth at 1000g for 10 minutes
  • Add 5 µl of the supernatant to 50μl water in a 1,5 ml tube and mix
  • deproteinize: Add 12.5μl of deproteinizing agent 1 and 2
  • incubate at 20°C for 15 minutes and centrifuge at 8000g for 5min
  • Transfer 50μl of supernatant in an extra test tube, include water blanks and standards
  • Add 50μl Color Agent to each and mix
  • Add 500μl of 32% v/v Hcl to each sample and mix
  • Heat for 20min at 50°C
  • Cool on Ice for 15min
    • Measure at 470nm using water as blank

PCR

  • 200ng template
  • 2μl forward primer (10μM)
  • 2μl reverse primer (10μM)
  • 1μl Q5 polymerase
  • 1μl dNTPs (10mM)
  • 10μl 5x Buffer
  • to 50μl with H2O
  • Mix components
  • Run thermocycler:
  • 98°C 2:00min
  • 98°C 0:30min
  • 55°C 0:45min
  • 72°C 2:00 min
  • 72°C 5:00 min
  • 4°C inf

Materials

50x TAE buffer

  • 121g Tris base
  • 28.5ml Acetic acid
  • 9.3g EDTA
  • to 500ml with H2O

LB

  • 25g Lennox Broth
  • to 1l with H2O

LB-Agar

  • 25g Lennox Broth
  • 15g Agar
  • to 1l with H2O

MRS medium

Medium prepared as described by manufacturer

antibiotics concentration

  • Chloramphenicol 34µg/ml
  • Ampicillin 100µg/ml

Transformation buffer for L. johnsonii

  • 342.3g Sucrose
  • 0.277g CaCl2
  • to 1l H2O