• 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

• 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

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

• 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

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

• 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

• 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

• 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