Team:Calgary/Troubleshooting



General Wet Lab

  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. To a microcentrifuge tube, add:

  1. To a microcentrifuge tube, add:
    • Digested vector DNA (in appropriate ratio)
    • Digested insert DNA (in appropriate ratio)
    • 1/10 of total final volume of aliquoted 10X T4 DNA ligase buffer
    • 1μl of T4 DNA ligase (1U/μl)
    • ddH2O to final volume
  2. Incubate at room temperature for 2 hours
  3. Transform chemically competent cells with the ligation product.
  4. Leave remaining ligation product at room temperature overnight, and transform again the following day.
  5. Store remaining product at -20°C

  1. Add 10μl of ddH2O to the desired well of the distribution kit plate
  2. .Pipette up and down 3-5 times (until solution becomes red)
  3. Incubate at room temperature for 10 minutes
  4. Transform cells with 1μl of rehydrated DNA as per transformation protocol

  1. Combine in a 0.2ml microcentrifuge tube:
    • 5μl of NEB 10X standard Taq buffer (final concentration = 1X)
    • 0.25μl of NEB Taq (final concentration = 1.25U/50μl)
    • 1μl of 10μM forward primer (final concentration = 0.2μM)
    • 1μl of 10μM reverse primer (final concentration = 0.2μM)
    • 1pg to 1ng of template plasmid DNA (final concentration < 1000ng/μl)
    • 1μl of 10mM Kapa dNTPs (final concentration = 200μM)
    • ddH2O to 50μl
  2. Vortex 2 to 3 seconds to mix, then centrifuge briefly to settle
  3. 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 (45 to 68°C) for 15 to 60 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 10μl of ddH2O to the desired well of the distribution kit plate
  2. .Pipette up and down 3-5 times (until solution becomes red)
  3. Incubate at room temperature for 10 minutes
  4. Transform cells with 1μl of rehydrated DNA as per transformation protocol

  1. To a microcentrifuge tube, add:
    • Required amount of DNA to be digested (can use NEBioCalculator to determine amount if performing a ligation with digested DNA)
    • 1/10 final total volume of appropriate 10X buffer
    • 1μl of each restriction enzyme (diluted)
    • ddH2O to final volume
  2. Incubate at 37°C (or other optimal temperature for enzyme activity) for 30 minutes to 3 hours (be careful of star activity)
  3. Heat inactivate restriction enzymes at 82°C for 20 minutes
  4. For digest confirmations, run on regular agarose gel. For subsequent ligation, run on LMP gel and excise or gel-extract. Otherwise, store at -20°C

E. coli Protocols

  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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Yeast Protocols


We would like to thank Dr. Vanina Zaremberg, Maria Laura Sosa Ponce, and Dr. Rodrigo Ledesma Amaro for graciously providing us with these protocols for Saccharomyces cerevisiae and Yarrowia lipolytica .


Reagents:

  • 6.7g Bactoyeast Nitrogen base without amino acids, with ammonium sulphate
  • 20g sugar (typically glucose)
  • 2.0mL 1% L-tryptophan
  • 2.0mL 1% L-methionine
  • 2.0mL 1% L-arginine
  • 2.0mL 1% L-leucine
  • 2.0mL 1% L-lysin
  • 10mL 0.2% Adenine sulfate
  • 10mL 0.2% Uracil

  1. In 500mL of ddH2O dissolve:
    • Bactoyeast nitrogen base (TNB, final concentration= 0.67% w/v)
    • Sugar (final concetration= 2% w/v)
  2. Under aseptic conditions add:
    • Amino acids from AUTOCLAVED 1% solutions
    • Bases from AUTOCLAVED 0.2% solutions

    *Add the amino acids and bases in the oder listed above to fill the desired auxotrophies. For example, to make SD-Ura (media lacking uracil), add all the amino acids and adenine, but not uracil.

  3. Bring volume to 1000mL with ddH2O, accounting for the volume(s) of amino acid and base solutions added.
  4. FOR PLATES: Add agar to final concentration of 2% (i.e 20g in 1L of media)

You can either:

  • Resuspend cells from a fresh plate in 1mL sterile 15% glycerol
  • Add 600uL of log phase culture (overnight culture) to 400uL of sterile 50% glycerol- vortex the culture first.

Work under aseptic conditions and store the tubes in the -80ºC freezer

  1. Vortex the starter tubes which were grown the day before.
  2. Prepare cuvettes in a 1:10 dilution and measure OD for each starter. The linear range of the measurement device is 0.5-1. lf other results are received then dilute your sample further. How to use the OD device? Press "L" so the reference stand (the blue stand) will move to the scanning place -> insert all cuvettes, starting with the reference one -> press "F2" -> press OK (the reference has been set) -> continue pressing OK for measuring.
  3. Make new starters that will reach mid-log (at this phase, the yeast cells have a thin cell wall, so it's easier to transform them with foreign DNA) according to the following principles: Cell cycle in YPD is 90 minutes and in SD is 120 minutes. You should calculate for a total of 0.4 OD after the waiting- at least 6h in SD and 4.5h in YPD. lt takes 2 divisions in order to be in mid log with no matter what is the measured OD. When the cells reach 1 OD they finish up all the sugar in the medium and go through diauxic shift. Since it takes one cell cycle for them to start dividing (in the first 90 minutes in YPD or L2O minutes in SD their OD remains exactly the same after back dilution) then at LEAST 3 cell cycles are needed to reach mid log.

  4. Place the new starters at 30oC until they reach logarithmic phase according to your plan. Measure the OD again (this time no need to dilute, just place 750 pl from the starters to the cuvettes.

    Important: steps 3+4 are only performed when you transform genomic DNA and want it to be integrated into the genome. Homologous recombination is obligatory in order to achieve this goal and it only takes place during cell cycle. lf you transform a plasmid then it is possible to use stationary phase yeast because the plasmid only needs to insert the nucleus and homologous recombination is not required.

  5. Make up sufficient Transformation Mix for the planned number of transformations plus one extra. It is not necessary to keep the transformation mix in ice. Once the Salmon Sperm's DNA has been boiled and cooled quickly it no longer has the energy to find its complementary strand. In addition, once you dilute it nearly 10 fold in the transformation mix, the chances of it complementing are really small so no problem leaving it outside the ice. Also the PEG is so viscous that there is not a lot of movement of the DNA (unlike in water).
    6. Reagents 1 5 (6X) 10 (11X)
    PEG 3500 50% w/v Mark Otto @mdo
    Lithium Acetate 1.0 M Jacob Thornton @fat
    Boiled Salmon Sperm-carrier of DNA Larry the Bird @twitter
    Plasmid/Genomic DNA plus Water Larry the Bird @twitter

Solution Preparation:

  1. Lithium Acetate (LiAc) Solution: 0.1M, pH 6.0
    • 5.1g LiAc for 500mL
    • Adujst to pH 6.0 with 10% acetic acid
    • Autoclave and store in the fridge
  2. Polyethylene Glycol (PEG) Solution: 40% PEG 4000 in 0.1M LiAc, pH 6.0
    • Add 50mL 0.1M LiAc to 40g PEG 4000
    • Adjust to pH 6.0 with 10% acetic acid
    • Adjust volume to 100mL with LiAc solution
    • Store in the fridge
  3. DNA Carrier: 5mg/mL fish DNA (salmon sperm)
    • 50mg DNA sodium salt/fish DNA
    • Dissolve in 10mL sterile TE buffer
    • Aliquot samples and store in -20ºC freezer

Part I: Preparation of Competent Cells

  1. Spread the strain that you want to transform on a YPD plate (Grow at 28°C for 16-24 hours)
  2. Put a loop of cells into 1ml of TE in sterile Eppendorf tubes
    • Prep for 5 transformations- i.e. 5 loops in 5 ml of TE
  3. Centrifuge 1 min at 10 000 rpm and remove supernatant
  4. Resuspend cell gently in 600µL of 0.1M LiAc pH 6.0
    • Prep for 5 transformations- i.e. 3mL
  5. Place in 28°C water bath without agitation for 1 hour
  6. Centrifuge for 2 min at 3000 rpm and remove supernatant
  7. Gently resuspend cells in 60µL of 0.1M LiAc pH 6.0
    • Prep for 5 transformations- i.e. 300µL

Part II: Transformation

  1. In a new eppendorf tube, mix together the following with a pipette:
    • 3-5μl of carrier DNA
    • 2-5µL your desired DNA
    • 40µL competent cells
  2. Incubate for 15 min in 28°C water bath without agitation
  3. Add 350µL PEG solution
  4. Incubate for 1 h in 28°C water bath without agitation
  5. Thermal shock the cells for 10 min at 39°C
  6. Add 600µL 0.1M LiAc pH 6.0
  7. Spread 200μl (or more) per plate containing the adequate selection medium

  1. Dissolve the following reagents in 1L of distilled water
    • 10g Yeast extract
    • 20g Peptones
    • 20g Dextrose
    • 20g Agar powder (FOR PLATES ONLY)
  2. Autoclave and allow media to cool slightly
  3. Add antibiotic/selection marker
  4. Store broth in the fridge OR aseptically pour into plates, swirl to remove bubbles, let set, and then store in the fridge upside-down.
NOTES:
  • Yeast extracts = contain vitamins, growth factors, minerals, or digested nucleic acids needed for growth.
  • Peptones = protein degradation productions (source of amino acids and nitrogen)
  • Dextrose = biochemically identical to glucose

*For other sugar media, REPLACE Dextrose with 20g of your desired sugar


Cellulase Integration

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Thymol 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.