Difference between revisions of "Team:Calgary/Experiments"

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<div class = "General Wet Lab: E.coli Protocols " id="general-wet">
 
<div class = "General Wet Lab: E.coli Protocols " id="general-wet">
 
          
 
          

Revision as of 08:03, 3 October 2020



General Wet Lab: E. coli Protocols

  1. Mix 100ml of 1X TAE buffer with 1g of regular or LMP agarose (for a 1% gel). For a small gel, mix 30ml of 1X TAE with 0.3g agarose.
  2. Microwave covered for about 1.5 minutes or until agarose is dissolved and pour into a balanced casting tray. LMP gels should be cast in the fridge.
  3. Add a volume of Safe-Red dye to be 1/6th the total volume of the sample.
  4. Run samples at 100V (regular gel) or 80V (LMP gel) until sample is ¾ of the way down the gel.

General Wet Lab: E. coli Protocols

  1. Mix 100ml of 1X TAE buffer with 1g of regular or LMP agarose (for a 1% gel). For a small gel, mix 30ml of 1X TAE with 0.3g agarose.
  2. Microwave covered for about 1.5 minutes or until agarose is dissolved and pour into a balanced casting tray. LMP gels should be cast in the fridge.
  3. Add a volume of Safe-Red dye to be 1/6th the total volume of the sample.
  4. Run samples at 100V (regular gel) or 80V (LMP gel) until sample is ¾ of the way down the gel.

  1. Add ≤ 5μl (≤ 1/10 of the cell aliquot amount) of the DNA sample to a chemically competent cell aliquot. Mix by pipetting gently, then incubate on ice for 30 to 45 minutes.
  2. Heat shock at 42°C for 1 minute
  3. Incubate on ice for 5 minutes
  4. Add 250μl of plain LB or SOC media aseptically, then incubate for 30 to 90 minutes at 37°C, shaking. *If resistance is Kan, must incubate for at least 1 hour
  5. Plate 100μl (if big plates) or 50μl (if small or half plates) aseptically
  6. Incubate plate at 37°C overnight or until growth is observed

*If transformation fails:

  • Spin down transformed cells for 5 minutes to pellet
  • Resuspend pellet in 100μl of media
  • Plate and incubate

  1. Culture overnight culture (O/N) in 2ml LB at 28°C, shaking
  2. Subculture (1:50) by adding 1ml O/N culture to 50ml LB with 10mM MgSO4 (500μl of 1M MgSO4) and 1mM KCl (50μl of 1M KCl)
  3. Shake at 28°C to OD600 = 0.3 to 0.4
  4. Chill on ice for at least 10 minutes
  5. Put into 50ml pre-chilled Falcon tube and centrifuge at 2500g for 8 minutes at 4°C (3450rpm in Allegra X-12 centrifuge)
  6. Resuspend in 10ml ice-cold 100mM CaCl2, gently mix on ice, then ice for at least 10 minutes
  7. Centrifuge at 2500g for 8 minutes at 4°C
  8. Resuspend in 500μl 100mM CaCl2 with 10% glycerol on ice, then incubate on ice for 10 minutes
  9. Aliquot 50μl into pre-chilled 1.5ml microcentrifuge tubes on ice. Store at -80°C
  10. Perform a test transformation with a reporter gene (i.e RFP)

  1. Create cPCR mastermix as follows:
    • 20μL 10X Taq Buffer
    • 4μL 10μM VF2 primer (or other forward primer)
    • 4μL 10μM VR primer (or other reverse primer)
    • 4μL 10mM dNTPs
    • 1μL Taq Polymerase
    • 127μL ddH2O

    *Each mastermix aliquot is enough to run 10 cPCR reactions/screen 10 colonies

  2. Add 4μL of ddH2O to each PCR tube or well (of a 96-well plate)
  3. Using pipette tips or sterile toothpicks, touch an individual labelled colony, swirl in the ddH2O, and then streak on masterplate. Repeat for as many colonies as desired.
  4. Add 16μL of cPCR mastermix to each tube/well
  5. Place in thermocycler and select or set up the appropriate program as follows:
    • Initial denaturation → 95°C for 5 minutes
    • Repeat 30x:
      • Denaturation → 95°C for 30 seconds
      • Annealing → Tm - 5°C (For VF2/VR primers, 53°C) for 30 seconds
      • Extension → 68°C for 1 minute per kilobase
      • Final extension → 68°C for 5 minutes
    • Final extension → 68°C for 5 minutes
    • Hold at 4°C

  1. Add 500μl of 50% glycerol to a 1.5ml tube aseptically
  2. Add 500μl of 50% of overnight culture to the tube aseptically. Mix gently
  3. Store at -80°C

  1. Mix into 1L of dH2O:
    • 10g of Tryptone (1%)
    • 5g of yeast extract (0.5%)
    • 10g NaCl (1%)
    • 15g Agar (1.5%)
  2. Autoclave and cool
  3. Add necessary antibiotics and mix thoroughly
    • 1ml/L Ampicillin (stock = 100mg/ml, final = 100μg/ml)
    • 1ml/L Kanamycin (stock = 50 mg/ml, final = 50μg/ml)
    • 0.5ml/L Chloramphenicol (stock = 50 mg/mlEtOH, final = 25μg/ml)
  4. Pour into plates aseptically, swirl to remove bubbles, and let set. Store in fridge upside-down.

  1. Mix into 1L of dH2O:
    • 10g of Tryptone (1%)
    • 5g of yeast extract (0.5%)
    • 10g NaCl (1%)
  2. Autoclave and cool
  3. Add necessary antibiotics and mix thoroughly
    • 1ml/L Ampicillin (stock = 100mg/ml, final = 100μg/ml)
    • 1ml/L Kanamycin (stock = 50 mg/ml, final = 50μg/ml)
    • 0.5ml/L Chloramphenicol (stock = 50 mg/mlEtOH, final = 25μg/ml)
  4. Pour into plates aseptically, swirl to remove bubbles, and let set. Store in fridge upside-down.
  5. Aliquot as necessary

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Anim pariatur cliche reprehenderit, enim eiusmod high life accusamus terry richardson ad squid. 3 wolf moon officia aute, non cupidatat skateboard dolor brunch. Food truck quinoa nesciunt laborum eiusmod. Brunch 3 wolf moon tempor, sunt aliqua put a bird on it squid single-origin coffee nulla assumenda shoreditch et. Nihil anim keffiyeh helvetica, craft beer labore wes anderson cred nesciunt sapiente ea proident. Ad vegan excepteur butcher vice lomo. Leggings occaecat craft beer farm-to-table, raw denim aesthetic synth nesciunt you probably haven't heard of them accusamus labore sustainable VHS.

Anim pariatur cliche reprehenderit, enim eiusmod high life accusamus terry richardson ad squid. 3 wolf moon officia aute, non cupidatat skateboard dolor brunch. Food truck quinoa nesciunt laborum eiusmod. Brunch 3 wolf moon tempor, sunt aliqua put a bird on it squid single-origin coffee nulla assumenda shoreditch et. Nihil anim keffiyeh helvetica, craft beer labore wes anderson cred nesciunt sapiente ea proident. Ad vegan excepteur butcher vice lomo. Leggings occaecat craft beer farm-to-table, raw denim aesthetic synth nesciunt you probably haven't heard of them accusamus labore sustainable VHS.

Anim pariatur cliche reprehenderit, enim eiusmod high life accusamus terry richardson ad squid. 3 wolf moon officia aute, non cupidatat skateboard dolor brunch. Food truck quinoa nesciunt laborum eiusmod. Brunch 3 wolf moon tempor, sunt aliqua put a bird on it squid single-origin coffee nulla assumenda shoreditch et. Nihil anim keffiyeh helvetica, craft beer labore wes anderson cred nesciunt sapiente ea proident. Ad vegan excepteur butcher vice lomo. Leggings occaecat craft beer farm-to-table, raw denim aesthetic synth nesciunt you probably haven't heard of them accusamus labore sustainable VHS.

TEF1 Promoter Testing

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Anim pariatur cliche reprehenderit, enim eiusmod high life accusamus terry richardson ad squid. 3 wolf moon officia aute, non cupidatat skateboard dolor brunch. Food truck quinoa nesciunt laborum eiusmod. Brunch 3 wolf moon tempor, sunt aliqua put a bird on it squid single-origin coffee nulla assumenda shoreditch et. Nihil anim keffiyeh helvetica, craft beer labore wes anderson cred nesciunt sapiente ea proident. Ad vegan excepteur butcher vice lomo. Leggings occaecat craft beer farm-to-table, raw denim aesthetic synth nesciunt you probably haven't heard of them accusamus labore sustainable VHS.

Cellulase Secretion

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Omega-3 Production

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Biocontainment

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Anim pariatur cliche reprehenderit, enim eiusmod high life accusamus terry richardson ad squid. 3 wolf moon officia aute, non cupidatat skateboard dolor brunch. Food truck quinoa nesciunt laborum eiusmod. Brunch 3 wolf moon tempor, sunt aliqua put a bird on it squid single-origin coffee nulla assumenda shoreditch et. Nihil anim keffiyeh helvetica, craft beer labore wes anderson cred nesciunt sapiente ea proident. Ad vegan excepteur butcher vice lomo. Leggings occaecat craft beer farm-to-table, raw denim aesthetic synth nesciunt you probably haven't heard of them accusamus labore sustainable VHS.