Team:WashU StLouis/Experiments

Experiments

Check out the protocols we used throughout our project

Cloning

We used these protocols to create plasmids with our genes of interest from genomic DNA, and transform those plasmids into E. coli


  1. Centrifuge incoulated cell culture for 15 minutes at 5000g
  2. Discard supernatant and resuspend in 1 mL of cloning water
  3. Transfer solution into bashing bead lysis/filtration tube
  4. Add 6 mL of fungal/bacterial DNA binding buffer
  5. Vortex for 5 minutes
  6. Spin down for 5 minutes at 5000g
  7. Transfer filter to a 50 mL tube
  8. Put spin column in collection tube and spin in a microcentrifuge for 1 minute at 10,000g
  9. Add 150 mL of DNA pre-wash buffer, spin for 1 minute at 10,000g, discard flow through, and repeat once
  10. Add 200 μL of fungal/bacterial DNA wash buffer to column, spin for 1 minute at 10,000g, and repeat 3 more times
  11. Transfer column to a 1.5 mL microcentrifuge tube and add 30 μL of cloning water
  12. Let sit for 4 minutes, then spin for 4 minutes at 10,000g
  13. Measure concentration of gDNA and label tube
  1. Add the following to a single PCR tube:
    • 20-21.5 μL Cloning Water (depending on type of amplicon)
    • 10 μL of Betaine
    • 10 μL of 5x High-Fidelity Buffer
    • 4 μL DMSO
    • 2.5 μL 10 uM combined forward/reverse primers
    • 1 μL DNTPs
    • 0.5 μL of plasmid or 2 μL of gDNA
    • 0.5 μL of Phusion Polymerase (kept on ice)
  2. Immediately run in a thermocycler for the following times/temperatures
    • Initial Denaturation 98°C 30 sec
    • 25-35 cycles
      • 98°C 5-10 sec
      • 45-72°C (at annealing temp) 10-30 sec
      • 72°C 15-30 sec/(length of amplicon in kb)
    • Final Extension 72°C 5-10 minutes
    • Hold at 4°C
  3. Store at 4°C until use
  1. Measure 1 gram of Agarose per 100 mL TAE buffer (Tris base, acetic acid and EDTA)
    • We generally made gels with 75 - 125 mL of TAE
  2. Microwave in a covered erlenmeyer flask for 120 seconds
  3. Add 1 μL of SYBR Safe or Ethidium Bromide per 20 mL of TAE
  4. Pour into gel casing
  5. Remove bubbles with a pipette tip
  6. Insert well comb
  7. Allow to set (~20 minutes)
  8. Remove well comb
  9. Place gel in electrophoresis apparatus with the wells by the cathode (black)
  10. Fill apparatus with TAE buffer so that the gel is completely submerged
  11. Load wells with 10-60 μL of 5 (PCR product): 1 (6x Loading Dye)
  12. Load 10 μL of ladder into first and last wells
  13. Run at 125-130 V for 25-30 minutes

(Adapted from Zymo Research Zymoclean™ Gel DNA Recovery Kit)

  1. Cut band from gel while minimizing time under UV light
  2. Place gel in a microcentrifuge tube with 3x volume of Agarose Dissolving Buffer
  3. Heat at 55°C until gel dissolves (~20 minutes)
  4. Add to gel purification filter placed inside a collection tube
  5. Centrifuge for 1 minute at 16,000 g and discard flow-through
  6. Add 200 μL of DNA wash buffer, centrifuge for 1 minute at 16,000 g, discard flow-through, and repeat once
  7. Centrifuge for 2 minutes at 16,000 g and discard flow-through
  8. Place filter in 1.5 mL microcentrifuge and add 20-30 μL of cloning water
  9. Wait for 4 minutes, then spin for 4 minutes at 16,000 g
  10. Measure concentrations and label

(Adapted from Zymo Research DNA Clean & Concentrator™-5 Kit)

  1. Add 50 μL DNA and 250 μL DNA Binding Buffer to a spin column in a collection tube
  2. Centrifuge for 30 seconds at Xg and discard flow-through
  3. Add 200 μL of DNA wash buffer, centrifuge for 30 seconds at 16,000g, discard flow-through, and repeat once
  4. Place filter in 1.5 mL microcentrifuge and add 20-30 μL of cloning water
  5. Wait for 4 minutes, then spin for 4 minutes at 16,000g
  6. Measure concentrations and label
  1. In a PCR tube, combine:
    • 0.4 μL Cutsmart Buffer
    • 0.4 μL DpnI
    • plasmid backbone (either post PCR or gel extract)
  2. Incubate at 37°C for 1 hour
  3. DNA purification for post-DpnI mix to retrieve plasmid backbone
  4. In a PCR tube, combine:
    • 100 ng of plasmid backbone
    • Equimolar amount of each assembly piece
    • 1.5 μL of restriction enzyme buffer
    • 1 μL of ligase buffer
    • 1 μL of Type IIs restriction enzyme
    • 1 μL of T4 ligase
    • Cloning water to equal a total of 15 μL
  5. Run in a thermocycler for the following times/temperatures:
    • 50 cycles
      • 3 minutes at 37°C
      • 4 minutes at 16°C
    • 1 cycle
      • 5 minutes at 50°C
      • 20 minutes at 80°C
    • Hold at 4°C
  6. Transform into competent cells and/or store at -20°C

Electroporation

  1. Defrost competent cells on ice for 5 minutes
  2. Pipet 1.5 μL plasmid to the tube of competent cells
  3. Pipet the mixture into an electroporation cuvette
    • Avoid touching the metal plate of the cuvette and introducing bubbles
  4. Shock
  5. Quickly add 500 μL of Lysogeny Broth (LB)
  6. Incubate in a culture tube for 1 hour at 37°C
  7. Pipet desired amount of solution onto a plate and spread evenly
  8. Allow solution to dry and incubate for 16-18 hours
  9. Wrap plate in parafilm and store at 4°C

Chemical Transdormation

  1. Thaw 100 μL of competent cells on ice
  2. Add 100 ng of plasmid to cells
  3. Incubate on ice for 20-30 minutes
  4. Heat shock the cells for 60 seconds at 42°C
  5. Return the cells to ice for 2 minutes
  6. Add 300 μL of LB or SOB medium
  7. Incubate in a culture tube for 1 hour at 37°C
  8. Pipet desired amount of solution onto a plate and spread evenly
  9. Allow solution to dry and incubate for 16-18 hours
  10. Wrap plate in parafilm and store at 4°C

Adapted from Zymo Research’s Zyppy Plasmid Miniprep kit

  1. Spin down cells for 15 minutes at 3200g
  2. Discard flow-through and resuspend in 600 μL of cloning water
  3. Transfer to a 1.5 mL microcentrifuge tube
  4. Add 100 μL of 6x Lysis Buffer and invert
  5. Add 300 μL of cold Neutralization Buffer and mix thoroughly
  6. Microcentrifuge for 4 minutes at 16,000g
  7. Pour supernatant into a miniprep filter placed in a collection tube
  8. Microcentrifuge for 1 minute at 16,000g and discard flow-through
  9. Add 200 μL of Endo-wash buffer
  10. Microcentrifuge for 30 seconds at 16,000g and discard flow-through
  11. Add 400 μL of Zyppy Wash buffer
  12. Microcentrifuge for 30 seconds at 16,000g and discard flow-through
  13. Microcentrifuge for 2 minutes at 16,000g and discard flow-through
  14. Place filter in 1.5 mL microcentrifuge and add 20-30 μL of cloning water
  15. Wait for 4 minutes, then spin for 4 minutes at 16,000g
  16. Measure concentrations and label
  1. Add 4 mL of Lysogeny broth (LB) and 4 μL of each needed antibiotic to a culture tube
  2. Add cells to the culture tube
    • If using frozen stock, scrape a small amount of stock with a pipette tip
    • If using a plate, carefully scrape a single colony with a pipette tip
  3. Grow at 37°C for 16-18 hours

BioBrick

We used these protocols to manipulate BioBricks provided in the iGEM distribution kit and to create our own BioBricks from experimentally tested genes


  1. Add the following to a PCR tube
    • 29 μL of miniprepped plasmid
    • 11 μL Cloning water
    • 5 μL of NEB Buffer (depends on REs used)
    • 5 μL total of Restriction Enzymes (SpeI, PstI, EcoRI, or XbaI)
  2. Set at 37°C for 3 hours
  3. Heat inactivate at 80°C for 20 minute
  1. Add the following to a PCR tube
    • 100ng of backbone
    • 3:1 ratio of insert:backbone
    • 2 μL 10X T4 DNA Ligase Buffer
    • 1 μL T4 DNA Ligase
    • Add Cloning water to get total volume of 20uL
  2. Set at room temperature (~23°C) for 1-3 hours
  3. Heat inactivate at 80°C for 20 minutes

Measurement and Assays

We used these protocols to test the effects of our plasmid constructs and quantify those results


Adapted from Thermo Fisher Scientific Molecular Probes® ATP Determination Kit

Link to Protocol

Adapted from Vector Laboratories Quant*Tag Biotin Quantification Kit

Link to Protocol

Miscellaneous

These protocols served a variety of important purposes


  1. Streak cells from frozen stock onto LB plate and incubate overnight at 37°C
  2. Pick a colony and inoculate in LB (with antibiotic if applicable) and incubate overnight at 37°C
  3. Dilute 1 mL of culture into 50 mL LBMgSO (with antibiotic if applicable) pre warmed to 37°C
  4. Grow culture in 37°C in a shaker until it reached an OD600 of 0.6 (2-5 hours depending on presence of antibiotic)
  5. Incubate cells for 20 minutes on ice
  6. Transfer the cells to ice-cold sterile 50 mL tube
  7. Centrifuge for 10 minutes at 3000 rpm at 4°C and discard the supernatant
  8. Gently resuspend the cells in 20 mL of ice-cold TFB1 with chilled pipet tips
  9. Incubate cells on ice for 25 minutes
  10. Repeat centrifuge, TFB1 resuspension, and 25 minutes on ice
  11. Resuspend cells in 4 mL of ice-cold TFB2
  12. Aliquot 100 mL cells into prechilled 1.5 mL microcentrifuge tubes and freeze immediately in liquid nitrogen
  13. Store at -80°C
  1. Place 7.5 g Agar and 12.5 g Powdered LB Broth in a jar
  2. Fill to 500mL mark with deionized water
  3. Add a magnetic stir bar and mix thoroughly using a stir plate
  4. Autoclave
  5. Let cool, add 500 μL of (each) antibiotic, and stir using stir plate
  6. In a biohood, Pipet 20-25 mL solution into labeled plates and allow to dry
  7. Store at 4°C
  1. Inoculate cell culture
  2. Combine 500 μL of culture with 500 μL of 30% glycerol in a labeled cryo tube
  3. Invert to mix and store at -80°C

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