Team:WashU StLouis/Experiments

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Protocols

Check out the protocols we used for our project



Cloning

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  • Centrifuge (X mL) cell culture for 15 minutes at 5000g
  • Discard supernatant and resuspend in 1 mL of cloning water
  • Transfer solution into bashing bead lysis/filtration tube
  • Add 6 mL of fungal/bacterial DNA binding buffer
  • Vortex for 5 minutes
  • Spin down for 5 minutes at 5000g
  • Transfer filter to a 50 mL tube
  • Put spin column in collection tube and spin in a microcentrifuge for 1 minute at 10,000g
  • Add 150 mL of DNA pre-wash buffer, spin for 1 minute at 10,000g, discard flow through, and repeat once
  • Add 200 uL of fungal/bacterial DNA wash buffer to column, spin for 1 minute at 10,000g, and repeat 3 more times
  • Transfer column to a 1.5 mL microcentrifuge tube and add 30 uL of cloning water
  • Let sit for 4 minutes, then spin for 4 minutes at 10,000 g
  • Measure concentration of gDNA and label tube

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  • Add the following to a single PCR tube:
    • 20-21.5 uL Cloning Water (depending on type of amplicon)
    • 10 uL of Betaine
    • 10 uL of 5x High-Fidelity Buffer
    • 4 uL DMSO
    • 2.5 uL 10 uM combined forward/reverse primers
    • 1 uL DNTPs
    • 0.5 uL of plasmid or 2 uL of gDNA
    • 0.5 uL of Phusion Polymerase (kept on ice)
  • Immediately run in a thermocycler for the following times/temperatures
    • Initial Denaturation 98C 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 4C
  • Store at 4C until use

Our iGEM team shared lab space in two different labs. Because of this, we learned two different protocols for gel electrophoresis:


Gel Electrophoresis with SYBR safe (Moon Lab)

Making the Gel

  • Measure 1 gram of Agarose per 100 mL TAE buffer (Tris base, acetic acid and EDTA)
    • We generally made gels with 75 - X mL of TAE
  • Microwave in a covered erlenmeyer flask for 120 seconds
  • Add 1 uL of SYBR Safe per 20 mL of TAE
  • Pour into gel casing
  • Remove bubbles with a pipettor
  • Insert well comb
  • Allow to set (~20 minutes)

Running the Gel

  • Remove well comb
  • Place gel in electrophoresis apparatus with the wells by the cathode (black)
  • Fill apparatus with TAE buffer so that the gel is completely submerged
  • Load wells with 10-60 uL of 5 (PCR product): 1 (6x Loading Dye)
  • Load 5-10(?) uL of ladder into first and last wells
  • Run at 125(?) V and 25(?) amps for 30(?) minutes

Gel Electrophoresis with Ethidium Bromide
  • Put
  • Things
  • Here

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

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Adapted from X kit
  • Add 50 uL DNA and 250 uL DNA Binding Buffer to a spin column in a collection tube
  • Centrifuge for 30 seconds at Xg and discard flow-through
  • Add 200 uL of DNA Wash Buffer, centrifuge for 30 seconds at Xg, discard flow-through,
  • and repeat once
  • Place filter in 1.5 mL microcentrifuge and add 20-30 uL of cloning water
  • Wait for 4 minutes, then spin for 4 minutes at 16,000 rcf?
  • Measure concentrations and label

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

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Adapted from Zymo Research’s Zyppy Plasmid Miniprep kit
  • Spin down cells for 15 minutes at 3200 rcf
  • Discard flow-through and resuspend in 600 uL of cloning water
  • Transfer to a 1.5 mL microcentrifuge tube
  • Add 100 uL of 6x Lysis Buffer and invert
  • Add 300 uL of cold Neutralization Buffer and mix thoroughly
  • Microcentrifuge for 4 minutes at 16,000 g
  • Pour supernatant into a miniprep filter placed in a collection tube
  • Microcentrifuge for 1 minute at 16,000 g and discard flow-through
  • Add 200 uL of Endo-wash buffer
  • Microcentrifuge for 30 seconds at 16,000g and discard flow-through
  • Add 400 uL of Zyppy Wash buffer
  • Microcentrifuge for 30 seconds at 16,000g and discard flow-through
  • Microcentrifuge for 2 minutes at 16,000g and discard flow-through
  • Place filter in 1.5 mL microcentrifuge and add 20-30 uL of cloning water
  • Wait for 4 minutes, then spin for 4 minutes at 16,000 rcf
  • Measure concentrations and label

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  • Add 4 mL of Lysogeny broth (LB) and 4 uL of each needed antibiotic to a culture tube
  • 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
  • Grow at 37 C for 16-18 hours

BioBrick

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Measurement and Assays

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Miscellaneous

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  • Streak cells from frozen stock onto LB plate and incubate overnight at 37 C
  • Pick a colony and inoculate in LB (with antibiotic if applicable) and incubate overnight at 37 C
  • Dilute 1 mL of culture into 50 mL LBMg (with antibiotic if applicable) pre warmed to 37 C
  • Grow culture in 37 C in a shaker until it reached an OD600 of 0.6 (2-5 hours depending on presence of antibiotic)
  • Incubate cells for 20 minutes on ice
  • Transfer the cells to ice-cold sterile 50 mL tube
  • Centrifuge for 10 minutes at 3000 rpm at 4 C and discard the supernatant
  • Gently resuspend the cells in 20 mL of ice-cold TFB1 (name) with chilled pipet tips
  • Incubate cells on ice for 25 minutes
  • Repeat centrifuge, TFB1 resuspension, and 25 minutes on ice
  • Resuspend cells in 4 mL of ice-cold TFB2
  • Aliquot 100 mL cells into prechilled 1.5 mL microcentrifuge tubes and freeze immediately in liquid nitrogen
  • Store at -80 C

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  • Place 7.5 g Agar and 12.5 g Powdered LB Broth in a jar
  • Fill to 500mL mark with Deionized water
  • Add a magnetic stir bar and mix thoroughly using a stir plate
  • Autoclave (settings?)
  • Let cool, add 500 uL of (each) antibiotic, and stir using stir plate
  • In a biohood, Pipet 20-25 mL solution into labeled plates and allow to dry
  • Store at 4 C

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  • Inoculate cell culture
  • Combine 500 uL of culture with 500 uL of 30% glycerol in a labeled cryo tube
  • Invert to mix and store at -80 C

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