Team:Calgary/Experiments

Notebook

Protocols

General Laboratory Protocols

LB Agar Plates
  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.
LB Liquid Broth
  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. Aliquot as required
SOB Media
  1. Per litter, add:
    • 950 mL deionized H2O, 20g tryptone, 5g yeast extract, 0.5g NaCl.
  2. Shake until dissolved. Add 10 mL of 250 mM KCl solution:
    • 250 mM KCl solution: dissolve 1.86g KCl in 100mL of deionized H2O.
  3. Adjust pH of medium to 7.0 with 5N NaOH (±0.2 mL).
  4. Adjust volume of solution to 1.0L with deionized H2O.
  5. Autoclave.
  6. Add 5mL of sterile 2M MgCl2 solution
    • 2M MgCl2 solution: dissolve 19g MgCl2 in 90 mL deionized H2O.
  7. Adjust volume of solution to 100mL with deionized H2O. Sterilize by autoclaving.

    8. Reference: Green, Michael R., and Joseph Sambrook. Molecular Cloning a Laboratory Manual. Cold Spring Harbor Laboratory Press, 2012.

SOC Media
  1. Autoclave SOB medium. Cool to 60 ̊C.
  2. Add 20 mL Sterile 1 M Glucose
    • Dissolve 18g glucose in 90 mL of deionized H2O
    • Adjust volume of solution to 100mL with deionized H2O
    • Sterilize by passing it through a 0.22μm filter

    Reference: Green, Michael R., and Joseph Sambrook. Molecular Cloning a Laboratory Manual. Cold Spring Harbor Laboratory Press, 2012.

Rehydration of Registry DNA
  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
Chemically Competent E. coli cells
  1. 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
Bacterial Transformations
  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
Plasmid Miniprep

P1 (Resuspension buffer) at 4°C on ice

  • 50mM Tris•HCl (pH = 8), 10mM EDTA, 100μg/ml RNase A

P2 (Lysis buffer) at room temperature

  • 200mM NaOH, 1% SDS

P3 (Precipitation buffer) at room temperature

  • 3M potassium acetate (pH = 5.5)

 

  1. Grow 2 to 6ml culture O/N
  2. Transfer 2ml (at a time) to 2ml microcentrifuge tube(s) and pellet at 14000rpm for 5 minutes. Discard supernatant and repeat as necessary
  3. Resuspend in 300μl ice-cold P1
  4. Add 300μl P2, gently invert 3x. Quickly add 300μl P3 and invert 3x
  5. Spin at 14000rpm for 10 minutes at room temperature
  6. Retain supernatant in 1.5ml microcentrifuge tube
  7. Add 650μl of 100% isopropanol, gently invert, and incubate for 10 minutes at room temperature
  8. Spin at 14000rpm for 10 minutes, then discard supernatant
  9. Wash pellet with 500μl of cold 70% ethanol
  10. Spin at 14000rpm for 5 minutes
  11. Discard supernatant. Carefully tap tube to remove remaining ethanol. Dry pellet in vacufuge
  12. Resuspend pellet in 20μl of sterile ddH2O or TE buffer. Nanodrop to determine concentration. Store at -20°C
Glycerol Stock
  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
Agarose Gel Electrophoresis
  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
  3. Cool and add 4μl SYBR safe. For a small gel, add 1.5μl SYBR safe.
  4. Set up gel tray and balance properly. Pour and cast gel. LMP gels should be cast in the fridge.
  5. Run sample at 100V (regular gel) or 80V (LMP gel) until sample is ¾ of the way down the gel.
Restriction Digest
  1. To a microcentrifuge tube, add:
    • Required amount of DNA to be digested
    • 1/10 final total volume of appropriate 10X buffer
    • 1μl of each restriction enzyme (diluted)
    • ddH2O to final volume
  2. Incubate at 37°C for 30 minutes to 3 hours
  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
DNA Excision from Low Melting Point Gel
  1. For a 1% gel, add 0.3g low melting point agarose to 30mL TAE buffer in a 250mL Erlenmeyer flask and microwave (covered) until agarose is fully dissolved
  2. Allow flask to cool until warm to the touch before adding 1.5μL RedSafe nucleic acid staining solution. Gently swirl to mix
  3. Pour agarose into assembled gel casting tray in the fridge. Remove any bubbles with a pipette tip and place comb in gel
  4. Allow gel to solidify and transfer to a gel running apparatus filled with TAE buffer
  5. Load samples of DNA containing 6X loading dye
  6. Run gel at 80V for 30 minutes or until loading dye is 2/3 way down the gel
  7. Remove the gel from casting tray and place it on a UV-light viewer. While using a UV-protecting face mask and shield, turn on the UV light and use razor blade to excise out the desired DNA bands (as quickly as possible as UV-light damages the DNA). Place each band into individual 1.5mL microcentrifuge tubes
  8. Melt the gel at 65°C for 5 minutes 
  9. Measure the volume of the excised gel slice. Add an equal volume of ddH2O to the tube and mix well. Determine the concentration of DNA and write on tube.
  10. Use in ligation or store at -20°C

*Gel-excised DNA samples may need to be re-heated to 65°C once again before ligation to desolidify

Ethanol/Salt DNA Precipitation
  1. To DNA sample, add:
    • 1/10 volume of 3M sodium acetate
    • 2 to 3 volumes of 100% ethanol
  2. Freeze at -80°C for 45 minutes
  3. Spin at max for 30 minutes at 4°C
  4. Discard supernatant Spin 5 minutes at 14,000g.
  5. Wash with 200-500 uL 70% ethanol
  6. Remove supernatant.
  7. Dry pellet in the vacufuge for 10-15 minutes.
  8. Resuspend in required amount of ddH2O and vortex gently.
  9. NanoDrop and record concentration
Ligation of Digested DNA
  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
Golden Gate Assembly
  1. To a microcentrifuge tube, add:
    • Insert and vector DNA samples (PCR products or in donor vectors) in 1:1 or 2:1 insert:vector ratio
    • 1μl of BsaI-HF v2 (2U/μl)
    • 1μl of T4 DNA ligase (2U/μl)(diluted)
    • 2μl of T4 DNA ligase buffer
    • ddH2O to final volume
  2. Place in thermocycler and select or set up the appropriate program as follows:
    • Repeat 25x:
      1. Digestion -> 42°C for 2 minutes
      2. Annealing -> 16°C for 5 minutes
    • Final digestion step -> 60°C for 10 minutes
    • Heat inactivation step -> 80°C for 10 minutes
PCR
  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 30 seconds
    • Repeat 25 to 30x:
      1. Denaturation -> 95°C for 15 to 30 seconds
      2. Annealing -> Tm - 5°C (45 to 68°C) for 15 to 60 seconds
      3. Extension -> 68°C for 1 minute per kilobase
    • Final extension -> 68°C for 5 minutes
    • Hold at 4°C
Colony PCR
  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

  1. Add 4μL of ddH2O to each PCR tube or well (of a 96-well plate)
  2. 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.
  3. Add 16μL of cPCR mastermix to each tube/well
  4. Place in thermocycler and select or set up the appropriate program as follows:
    • Initial denaturation → 95°C for 5 minutes
    • Repeat 30x:
      1. Denaturation → 95°C for 30 seconds
      2. Annealing → Tm - 5°C (For VF2/VR primers, 53°C) for 30 seconds
      3. Extension → 68°C for 1 minute per kilobase
    • Final extension → 68°C for 5 minutes
    • Hold at 4°C
Inducing with IPTG
  1. Add 50uL of 1.0M IPTG to the subcultures with optical densities between 0.4 to 0.6.
  2. Place in the shaking incubator for 8 hours at 37°C or 24 hours at 25°C
SDS-Page

Preparing Protein Samples

  1. Mix an aliquot of the protein sample with an appropriate amount of 4x SDS-PAGE loading buffer (200mM Tris-Cl pH 6.8, 8% SDS, 40% glycerol, 0.4% bromophenol blue) to get a final concentration of 1x SDS-PAGE loading buffer
    • Add 1% β-Mercaptoethanol if required
  2. Boil samples for 2 minutes and spin down

Bacterial Transformation

Running Gels

  1. Set up SDS-PAGE apparatus with the comb-side of the gels facing the inner chamber
  2. Fill inner chamber completely with 1x SDS-PAGE running buffer (1x Tris-Glycine, 0.1% SDS), and outer chamber halfway
    • 1x SDS-PAGE running buffer can be reused approximately 4-5 times
  3. Load standards and samples
  4. Run for 10 minutes at 100V, followed by 30 minutes at 180V

Staining Gels

  1. Remove gel from plates and place in a box with enough Coomassie Blue staining solution (0.25% Coomassie R-250, 40% methanol, 10% glacial acetic acid) to cover the gel. Shake at room temperature for 45 minutes
  2. Destain the gel by replacing the Coomassie Blue staining solution with destaining solution (40% methanol, 10% glacial acetic acid) and shaking at room temperature for 10-30 minutes
  3. Repeat destaining step as necessary until distinct bands can be visualized
  4. The gel can be stored in dH2O at room temperature for a few weeks
Spinach Chlorophyll Extraction
  1. Measure 20mL of 80% acetone and add 1g fresh spinach leaves without stems. Grind until crushed thoroughly.
  2. Centrifuge the solution with the crushed spinach at 3750 rpm, -10°C for 9 minutes.
  3. Transfer the supernatant to a falcon tube and cover with foil to protect against photosensitivity.
Pheophytinase Reactions
  1. Prepare cell lysate and eluted proteins (refer to Ni-NTA Column Chromatography protocol).
  2. If using cell lysate: In a centrifuge tube, add 100uL cell lysate, 100uL pheophytin solution, 10uL of 20mM EDTA, and 50 uL of the pheophytinase reaction buffer (25 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 0.1% Triton X-100).
  3. If using elution fractions: add 100uL elution fraction, 100uL pheophytin solution, 10uL of 20mM EDTA, and 50uL of the pheophtyinase reaction buffer.
  4. Place in the incubator or heating block at 35°C and leave overnight or as needed.
Ni-NTA Column Chromatography
  1. Make 2-6ml overnights
  2. In the morning, subculture 2:50 in 50ml or 100ml LB + appropriate antibiotic. Incubate at 37°C, shaking until OD600 reaches 0.3 - 0.6 (0.5 is optimal)
  3. Induce with 100mM IPTG (50μl of 1M IPTG into 50ml, 100μl of 1M IPTG into 100ml) and incubate for 8-12 hours at 37°C or 16 hours at 30°C
  4. Centrifuge on max for 5 minutes and discard supernatant (x2 if 100ml culture). Freeze pellet
  5. Conduct 3x 20 minute freeze-thaw cycles for the pellet at -80°C and 37°C 
  6. Resuspend in 10 ml Resuspension/Equilibration buffer (1X PBS pH 7.4, 300mM NaCl, 10mM imidazole)
  7. Treat with 1mg/ml lysozyme (1ml of 10mg/ml lysozyme with 1% Tween80 into 10ml resuspension) for 30 minutes shaking at 37°C
  8. Transfer to 15ml falcon tube
  9. Sonicate (3x of 30A, 5 sec on, 5 sec off, 2 minutes total)
  10. At this point, you can take a 500μl sample to recover the soluble and insoluble whole cell lysate fraction if you so choose.
  11. Freeze sonicated samples for 20 minutes or overnight
  1. Take out required number of Ni-NTA columns and let them reach room temperature
  2. Take off top cap off and remove bottom plug. Place into a 50ml falcon tube and centrifuge at 700g for 5 minutes. Discard flowthrough
  3. Put 10ml of Resuspension buffer into the column and incubate for 10 minutes. Centrifuge at 700g for 5 minutes. Discard flowthrough and replace the bottom plug
  4. Load the 10ml of sonicated sample supernatant onto the columns and replace top cap. Incubate at 37°C, shaking for 30-60 minutes or longer.
  5. Label new 50ml falcon tubes with ‘post-loading sample’. Remove bottom plug and cap from the columns then place into the 50ml falcon tube. Centrifuge at 700g for 5 minutes and keep the flowthrough if desired.
  6. Perform 3 washes with 10ml Wash buffer (1x PBS pH 7.4, 300mM NaCl, 25mM imidazole). After each wash, centrifuge at 700g for 5 minutes and collect the wash samples if desired
  7. Add 4ml of Elution buffer (1x PBS pH 7.4, 300mM NaCl, 250mM imidazole) to the column, and incubate for at least 5 minutes. Centrifuge at 700g for 5 minutes and label collected fraction “Elution fraction 1”
  8. Repeat addition of 4ml of Elution buffer to the column, and incubate for at least 5 minutes. Centrifuge at 700g for 5 minutes and label collected fraction “Elution fraction 2”
  9. Repeat addition of 4ml of Elution buffer to the column, and incubate for at least 5 minutes. Centrifuge at 700g for 5 minutes and label collected fraction “Elution fraction 3”
  10. Wash column with 50mlo of MES buffer (20mM MES, 100mM NaCl). Centrifuge at 700g for 5 minutes (or until all buffer is through) and discard flowthrough
  11. Wash column with 50ml of nanopure water. Spin at 700g for 5 minutes (or until all water is through) and discard flowthrough
  12. Add about 30ml of 20% ethanol to the column and place back into the fridge. 
  13. Eluted samples can be used immediately or stored at 4°C or -20°C
Periplasmic Protein Isolation
  1. Make 2ml O/Ns and incubate at 37°C, shaking
  2. In the morning, subculture 2:50 in LB + appropriate antibiotic. Incubate at 37°C, shaking until OD600 reaches 0.3 - 0.6 (0.5 is optimal)
  3. Induce with 100mM IPTG (50μl of 1M IPTG into 50ml) and incubate for 8-12 hours at 37°C or 16 hours at 30°C
  4. Transfer to 50ml falcon tubes and centrifuge for 10 minutes at max. Discard supernatant
  5. Resuspend pellet in 10ml of 30mM Tris-HCl pH 8 + 20% sucrose
  6. Incubate for 10 minutes at room temperature
  7. Centrifuge for 10 minutes at max. Carefully discard supernatant
  8. Resuspend the pellet in 500μl of ice (fridge)-cold 5mM MgSO4 solution and transfer the suspension to a 1.5ml microcentrifuge tube
  9. Incubate the suspension for 30 minutes on ice
  10. Pellet the cells at 13000rpm for 10 minutes
  11. Transfer supernatant (shock fluid) to a fresh 1.5ml labelled microcentrifuge tube. This sample can now be visualized using SDS-PAGE, used in column chromatography, or stored at -20°C

If cytoplasmic preparation also desired:

  1. Resuspend pellet in 800μl of 1X PBS pH 7.2
  2. Add 80μl of 10mg/ml lysozyme with 1% Tween80. Incubate at 37°C for 0.5 hour
  3. Sonicate a sample of this solution before use (optional)
  4. Freeze samples 10-15 mins and boil 5 mins before sonication
Western Blot
  1. Run SDS PAGE gel
  2. Cut out PVDF membrane (also cut a corner to mark bottom and upper)
  3. Activate PVDF membrane with methanol by incubating for 15 sec
  4. Soak activated membrane in transfer buffer (1x Tris-glycine, 20% methanol),  to equilibrate for 5 min
    1. Do not let the membrane dry out! If a white spot is visible, rewet membrane in methanol first and then equilibrate in the transfer buffer
  5. Rinse gel in transfer buffer, wet filter paper and pads in the transfer buffer
  6. Assemble the sandwich in the transfer cassette as follows:
    1. White side of cassette (+)
    2. Sponge
    3. Whatmann filter paper (1 piece)
    4. PVDF membrane
    5. SDS Gel
    6. Whatman filter paper (1 piece)
    7. Sponge
    8. Black side of Cassette (-)
  7. Insert the cassette into the slot with black to balck and white to red
  8. Cover to just over top wire with transfer buffer after adding ice pack in the slot. Put the apparatus in the ice
  9. Run at 100 V for 1-3 hours
  10. Remove membrane from the sandwich. Do not allow to dry out
  11. Block the membrane in blocking solution (1x PBS with 5% skim milk) for 1 hour at R/T with shake (or at 4 degrees overnight without shake)
  12. Pour off blocking solution. Add primary antibody in blocking solution (1x PBS with 5% skim milk, 0.1ug/mL primary antibody) and incubate for 1 hr with shake
  13. Wash 3x with wash buffer (0.1% Tween in 1x PBS, a.k.a. PBST), 5 min each with shake
  14. Add secondary antibody in blocking solution (1x PBS with 5% skim milk, 0.04ug/mL secondary antibody) and incubate for 1 hr
  15. Wash 3x with wash buffer (0.1% Tween in 1x PBS, a.k.a. PBST), 5 min each with shake; additional wash with ddH2O with shake
  16. Add ECL western blotting substrate: premix 1 mL of each solution (solutions 1 and 2 from ECL kit) in a falcon tube and add the mixed solution to the membrane by pipetting several times to make sure the membrane fully contacts with the solution. Incubate at R/T for 5 min.
  17. Run protocol at BIO-RAD ChemiDocTM station using protocol as follows: 
    1. blots>>>Chemi>>>manually set exposure time: 10, 30, 50, ….300 second until you see the band
Pheophytin Production
  1. Add HCl to chlorophyll solution until final HCl concentration is 20 mM.
  2. After 2 minutes, add NaOH until 20mM final concentration in solution to neutralize the solution.
    • For pheophytinase reactions, add enough NaOH and HCl until final pH is 8.00.
Thin Layer Chromatography
  1. Draw a line 2.0cm away from the base of the silica plate.
  2. Use P20 pipettes to drop/apply solution to be run, leaving at least 1 cm away from the next solution. Add dropwise and allow to dry before re-application. Allow the whole silica plate to dry before placing in reaction chamber.
  3. Pour solvent of interest (70:30 Methanol to Hexane ratio) in the reaction chamber. Apply paper towels or filter papers on the walls of the chamber and wet with solvent of interest to saturate the chamber.
  4. Place the silica plate in the chamber after 5 minutes or full saturation. Wait for solvent to travel up the plate until solvent front of desired distance is reached.
  5. Mark the solvent front with a pencil. Visualize under UV light.
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