Team:Fudan China/Protocol

On this page, we would like to share with you the protocols that we have been using over the competition. Here you can find the exact methods we use to generate our data and results.

Chemical transformation

Materials

  • LB broth
  • Ligation product or known plasmid
  • Competent E. coli
  • Ice
  • Selection plates

Methods

  • Thaw 100µL competent E. coli cells on ice for 10 minutes
  • Add:
    • 10 µl DNA from a ligation reaction mix or
    • 10-100ng DNA of a known plasmid
  • Carefully flick the tube 4-5 times to mix cells and DNA. Do not vortex.
  • Place the mixture on ice for 30 minutes. Do not mix.
  • Heat shock at exactly 42°C for 90 seconds. Do not mix.
  • Place on ice for 2 minutes. Do not mix.
  • Pipette 900 µl of room temperature LB media into the mixture.
  • Incubate at 37°C and 200-250 rpm for 30-60 minutes.
  • Spread:
    • For ligation reaction DNA: 100µl of each transformation reaction onto a selection plate. If the clonies were sparse, for the rest of 900 µL:
      • Pellet cells at 8000rpm for 3 minutes
      • Remove and dispense 800 µL of supernatant
      • Re-suspend cells by pipetting
      • Plate resuspended cells as above
    • For known plasmid: 10 & 100 µL of each transformation reaction onto a selection plate. If the clonies were sparse, for the rest of 890 µL:
      • Pellet cells at 8000rpm for 3 minutes
      • Remove and dispense 800 µL of supernatant
      • Re-suspend cells by pipetting
      • Plate resuspended cells as above
  • Incubate overnight at 37°C with plates upside down.

Growing overnight cultures

Materials

  • 3 mL LB broth
  • 3 μL antibiotic(1000×)
  • Pipette tips
  • Test tubes with caps

Methods

  • Overnight cultures were prepared under sterile conditions using a Bunsen burner
  • Add 3 mL liquid LB media into test tubes
  • Add 3 μL of appropriate antibiotic into the broth
  • Using the pipette tips, pick a single colony and inoculate the cultures by dipping the tip into the LB broth or by adding 50 μL stored cells
  • Put caps on the tubes and incubate overnight at 37°C shaking at 200-250 rpm

PCR From Plasmid DNA Template

Materials

  • 2x Phanta Master mix
  • 10 µM forward primer
  • 10 µM forward primer
  • PCR tube
  • sterile water
  • Plasmid DNA

Methods

  • In a PCR tube on ice, combine 1-10 ng of plasmid DNA or just 1 µL template, 2 µL of 10 µM forward primer, 2 µL of 10 µM reverse primer to a PCR tube on ice, 25 µL of 2x Phanta Mastermix, and sterile water up to 50 µL.
  • Gently mix the reaction
  • If necessary, collect the liquid to the bottom of the PCR tube by spinning briefly
  • Transfer the PCR tube from ice to a PCR machine preheated to 98°C to begin thermocycling

Thermocycling

The PCR machine should be set to run the following steps:

Denaturation Denaturation Annealing Elongation Elongation Hold
95℃ 98℃ 45-72℃ 72℃ 72℃ 20℃
5:00 0:15 0:10 1 kb/min 5:00 ∞
25-35 cycles

Preparation of LB Broth

Materials

  • 10 g Tryptone
  • 5 g Yeast Extract
  • 10 g Sodium Chloride
  • 1 L Purified Water

Methods

  • Add 10 g Tryptone, 5 g Yeast Extract and 10 g Sodium Chloride to 1 litre purified water
  • Autoclave

Preparation of LB Agar

Materials

  • 10 g Tryptone
  • 5 g Yeast Extract
  • 10 g Sodium Chloride
  • 20 g Agar
  • 1 L Purified Water

Methods

  • Add 10 g Tryptone, 5 g Yeast Extract, 10 g Sodium Chloride and 20 g Agar to 1 litre purified water
  • Autoclave

Preparation of Glycerol Stocks

Materials

  • 500 µl glycerol (80%)
  • 1000 µl overnight culture in LB

Methods

  • Add 500 µl glycerol (80%) to 1.8 mL cryogenic vial
  • Add 1000 µl overnight culture in LB
  • Store at -80°C

Ligation

Materials

  • Microcentrifuge tube
  • 1 µL T4 DNA Ligase
  • 1 µL 10X T4 DNA ligase buffer
  • 2.5 µL Vector Plasmid
  • 1.5 µL Insert DNA
  • Sterile water

Methods

  • Add vector plasmid, insert DNA, T4 DNA ligase and T4 DNA ligase buffer to the microcentrifuge tube
  • Make reaction up to 10 µL using sterile water
  • Incubate at room temperature for 30 - 60 min

Restriction Digestion (Test Digest and Assembly Digest)

Materials

  • Restriction Enzyme
  • 10X buffer
  • Plasmid DNA
  • Sterile water

Methods

  • Set up the reaction following the instruction below, depending on whether test digest or assembly digest is being performed.
    • For a Test digest (10 µL):
      • 5 µL Plasmid DNA
      • 0.5 µL Restriction Enzyme 1
      • 0.5 µL Restriction Enzyme 2
      • 1 µL 10× buffer
      • Add sterile water to 10 µL
    • For gene assembly(30 µL):
      • 15 µL Plasmid DNA
      • 1.2 µL Restriction Enzyme 1
      • 1.2 µL Restriction Enzyme 2
      • 3 µL 10X buffer
      • Add sterile water to 30 µL
  • Incubate digestion reaction at 37°C for 30 minutes-1 hour
  • Perform heat deactivation at 80°C for 20 minutes, if not running on a gel at the end of incubation.

DNA Gel Electrophoresis

Materials

  • Agarose Powder
  • TAE buffer
  • Gel mould
  • 5 µL EB(10000×)
  • Gel Tank
  • 3-5 µL DNA ladder
  • DNA loading buffer

Methods

  • Prepare 1% w/v solution of agarose powder in 1×TAE buffer (e.g. 1g agarose powder in 100 mL buffer) using a conical flask.
  • Heat the mixture until agarose is completely dissolved.
  • Add 5 µL EB to the solution.
  • Pour the solution into a gel mould.
  • Allows the solution to set (approx 15-20 minutes)
  • Transfer the agarose gel to a tank, remove the comb and apply:
    • 3-5 µL of the DNA ladder
    • DNA samples with the corresponding amount of DNA loading buffer (6X)
  • Run the gel for 20 minutes at 120V.

PCR Purification, Gel Extraction & Miniprep

  • PCR purification was performed according to the Generay PCR Purification Kit
  • Gel extraction was performed according to the Generay Gel Extraction Kit
  • Plasmid extraction were carried out according to the Generay Plasmid extraction Kit

DNA Gel Electrophoresis

Materials

  • 4 µL CE Ⅱ buffer
  • 2 µL Enzyme
  • 1 µL Vector Plasmid
  • 1 µL Insert DNA
  • 12 µL Sterile water

Methods

  • Add CE Ⅱ buffer, Enzyme, Vector Plasmid, Insert DNA to the microcentrifuge tube
  • Make reaction up to 10 µL using sterile water
  • Incubate at 37℃ for 30 min

Arabinose/Lactose Induction and Sampling

Materials

  • Arabinose/Lactose
  • 4 mL LB broth
  • 4 μL antibiotic(1000×)

Methods

  • Add antibiotic into LB borth to make antibiotic LB borth
  • Dissolve 0.1 g arabinose/lactose in 1 mL antibiotic LB borth and sterilize by filter sterilization
  • Add 850 μL antibiotic LB borth into 2 microcentrifuge tube each and inoculate the cultures by adding 50 μL stored cells each tube
  • Cultivate in 37℃, 200 rpm for over 2 hours
  • Sampling
    • Add 10 μL culture into 90 μL sterile water and start timing
    • Perform heat deactivation at 80°C for 20 minutes
  • Add 100 μL arabinose/lactose solution for test group, and 100 μL antibiotic LB borth for the control right after starting timing
  • Cultivate in 37℃, 200 rpm for exact time
  • Sampling

qPCR

Materials

  • 5 µL Aceq Mix
  • 0.2 µL forward primer
  • 0.2 µL reverse primer
  • 0.2 µL Reference dye
  • Template

Methods

  • Dilute the samples 10 times
  • Add 5 µL Aceq Mix, 0.2 µL forward primer, 0.2 µL reverse primer, 0.2 µL Reference dye and 4.4 µL Template into qPCR Tube and set 4 parallel groups for each sample
  • Avoid light when adding reagents

Thermocycling

The qPCR machine should be set to run the following steps:

Denaturation Denaturation Annealing and Elongation Annealing and Elongation
95℃ 95℃ 55℃ 95℃ 55℃ 95℃
10:00 0:10 0:34 1:00 0:30 0:30
1 cycle 40 cycles 1 cycle