Team:WashU StLouis/Experiments

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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


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  • Centrifuge incoulated 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 - 125 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 pipette tip
  • 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 10 uL of ladder into first and last wells
  • Run at 125-130 V for 25-30 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 g and discard flow-through
  • Add 200 uL of DNA wash buffer, centrifuge for 1 minute at 16,000 g, discard flow-through, and repeat once
  • Centrifuge for 2 minutes at 16,000 g 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 g
  • 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 16,000g, 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 g
  • Measure concentrations and label

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  • In a PCR tube, combine:
    • 100 ng of plasmid backbone
    • Equimolar amount of each assembly piece
    • 1.5 uL of restriction enzyme buffer
    • 1 uL of ligase buffer
    • 1 uL of Type IIs restriction enzyme
    • 1 uL of T4 ligase
    • Cloning water to equal a total of 15 uL
  • 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
  • Transform into competent cells and/or store at -20 C

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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
  • Thaw 100 uL of competent cells on ice
  • Add 100 ng of plasmid to cells
  • Incubate on ice for 20-30 minutes
  • Heat shock the cells for 60 seconds at 42 C
  • Return the cells to ice for 2 minutes
  • Add 300 uL of LB or SOB medium
  • 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

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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

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


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Measurement and Assays

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


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  • step 1
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  • step 1
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Miscellaneous

These protocols served a variety of important purposes


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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
  • 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