Team:Lambert GA/Experiments

EXPERIMENTS

CLONING PROTOCOL

RESTRICTION DIGEST

Reagent Amount
Restriction Enzyme 1 1 uL
Restriction Enzyme 2 1 uL
10x Cutsmart Buffer 5 uL
Insert or Vector DNA 1 ug
Nuclease Free Water/ Sterile Water To 50 uL
  1. Shave ice in styrofoam container
  2. Label PCR tubes
  3. Add all reagents to tube
  4. Incubate for 30 minutes at 37°C
  5. Incubate for 20 minutes at 65°C

LIGATION

Reagent Amount
T4 Ligase Buffer 2 uL
Vector DNA 50 ng
Insert DNA 150 ng
Nuclease Free Water To 20 ng
T4 DNA Ligase To 50 uL
  1. Shave ice
  2. Label all PCR tubes
  3. Add all of the reagents to your tube
  4. Incubate for 10 min at room temperature
  5. Incubate for 10 min at 65°C

TRANSFORMATION

  1. Label tubes with initials, date, and what is being transformed
  2. Place all tubes on ice
  3. Put SOC/LB and antibiotic plates in the incubator to warm at 37°C
  4. Pipette 1ul of the desired plasmid(s) and 1ul of PUC19 into the corresponding labeled transformation tubes
  5. Thaw competent cells on ice for 10 minutes or just until the cells inside are moving when you flick the tube
  6. Pipette 50ul of competent cells into each labeled transformation tube on ice
  7. Incubate tubes on ice for 30 minutes
  8. Heat shock tubes at 42°C for 45 seconds
  9. Put on ice 2 minutes
  10. Add 950ul of SOC/LB media into each tube with cells
  11. Place tubes shaking in the incubator for 60 minutes at 37°C
  12. Pipette 120-150ul of cells with SOC/LB media onto warm plates with the correct antibiotics
  13. Place antibiotic plates in the incubator at 37°C for 24 hours to let cells grow

INOCULATION FOR CULTURES

  1. Prepare Liquid LB
  2. When ready to grow culture, add Liquid LB to a tube or flask and add your antibiotic that your cells are resistant to the correct concentration to create a 1 milliliter of LB to 1 microliter of antibiotic (typically use 5 ml: 5 ul)
  3. Using a sterile pipette tip select a single colony from your LB agar plate
  4. Drop the tip into liquid LB and antibiotic, then swirl
  5. Cover culture with sterile aluminum foil or cap that is not airtight
  6. Incubate culture at 37℃ for 12-18 hours in a shaking incubator
  7. After incubation, check for growth
  8. For long term, storage continue with creating a glycerol stock

MINIPREP

  1. Grow 1-5 mL culture overnight in a 10mL-20mL culture tube.
  2. Centrifuge at 2500 xg for 10 minutes at room temperature. Decant or aspirate and discard culture media.
  3. Add 250 uL of solution 1 mixed with RNase. Vortex to mix, then transfer suspension into a new 1.5 mL microcentrifuge tube.
  4. Add 250 uL of solution 2. Invert until there is clear lysate.
  5. Add 350 uL to solution 3, insert until white precipitate forms. Centrifuge at 13,000 xg for 10 minutes.
  6. Insert mini column into a 2 mL collection tube.
  7. Transfer the clear supernatant into a mini-column using a micropipette. Centrifuge at 13,000xg for 60 seconds. Discard filtrate and reuse the collection tube.
  8. Add 500 uL of HBC Wash Buffer diluted in ethanol. Centrifuge at 13,000xg for 60 seconds. Discard filtrate and reuse the collection tube.
  9. Add 700 uL of the DNA Wash Buffer diluted in ethanol. Centrifuge at 13,000xg for 30 seconds, Discard filtrate, and reuse collection tube.
  10. Centrifuge empty mini-column at 13,0000xg for 2 minutes to remove ethanol.
  11. Transfer mini-column to a nuclease-free 1.5 mL microcentrifuge tube.
  12. Add 50 uL of Elution Buffer. Let it sit at room temperature for 5 minutes. Centrifuge at 13,000xg for 60 seconds.
  13. Store eluted DNA at -20℃

GEL ELECTROPHORESIS

MAKING GEL:

  1. Mix 0.5g of agarose with 50ml of 1x TAE buffer in a 100-250ml Erlenmeyer flask
  2. Heat up the solution until it turns clear
  3. Let the solution cool down until it reaches 65°C
  4. Once the solution measures 65°C, add 6ul of SybrSafe in and gently shake the flask to mix the solution
  5. Pour the solution into a gel chamber and put in the gel comb
  6. Wait for the gel to solidify before loading

RUNNING THE GEL:

  1. Once solidified, fix the gel’s position so that the wells of the gel are at the end of the chamber and DNA runs to red
  2. Pour used 1x TAE buffer into the gel chamber evenly to completely cover the gel
  3. On a piece of parafilm, mix 5ul of DNA and 1ul of purple loading dye using a micropipette
  4. Add the mixed 6ul solution into each well
  5. Add 6ul of 2-log ladder into an empty well
  6. Connect the electrodes by closing the gel chamber and connecting the to the power supply
  7. Set power supply for 90 volts and 45 minutes
  8. Turn on the power supply and make sure bubbles are rising on the sides of the gel chamber
  9. Once gel electrophoresis is completed, put the gel under UV (ultraviolet) light to compare the bands of DNA to the bands of the ladder

PCR

Reagent Amount
Sterile water To 25 uL (normally 9uL)
Q5 High Fidelity Master Mix 12.5 uL
10 uM diluted forward primer 1.25 uL
10 uM diluted reverse primer 1.25 uL
Template DNA 1 uL
  1. Add reagents to labeled PCR tube
  2. Set thermocycler with new annealing temperature and elongation times (annealing time and temperature depends on your part to PCR)
  3. Run PCR

GIBSON ASSEMBLY

PART 1: ASSEMBLY

Reagent Amount
 Sterile water To 20 uL
 Gibson assembly master mix (2x)  10 uL
 PCR fragments  Variable (dep on calculation done above - norm 1 uL)
 Linearized Vectors  Variable (dep on calculation done above - norm 1 uL)
  1. Add all reagents into labeled PCR tubes
  2. Add 10 uL Gibson positive control and 10 uL master mix to another labeled PCR tube for your control
  3. Incubate in thermocycler at 50 degrees C for 15 mins when assembling 2 or 3 fragments or 50 degrees C for 60 mins when assembling 4-6 fragments
  4. Store at -20 degrees C

PART 2: TRANSFORMATION

  • Add 2 uL of Gibson/ligated DNA to microcentrifuge tubes and 1 uL Puc into another tube
  • Add 50 uL competent cells to microcentrifuge tubes and mix by pipetting up and down 4-5 times
  • Place microcentrifuge tubes on ice for 30 minutes
  • Heat shock at 42 degrees for 30 seconds
  • Transfer tubes on ice for 2 minutes
  • Add 950 uL SOC media to tubes
  • Place tubes in shaking incubator at 37 degrees C for 60 mins
  • Plate 200 uL cells onto antibiotic plates (Gibson Positive Control goes on a carb plate)
  • Incubate plates overnight at 37 degrees C

    • COLONY PCR

    Reagent Amount
    Q5 High-Fidelity 2X Master Mix 12.5uL
    10uM Forward Primer VF2 1.25uL
    10uM Reverse Primer VR 1.25uL
    Template DNA 1.25 uL
    Nuclease-Free Water To 25uL
    1. Choose colony and dilute with 40uL of water
    2. Take 1uL out for the colony PCR
    3. Put all together in PCR tube
    4. Place in Thermocycler

    PCR CLEANUP

    1. Add 1:1 volume of binding buffer to completed PCR mixture
    2. Transfer up to 800 uL to GeneJET purification column
    3. Centrifuge for 60 seconds and discard flow through
    4. Add 700 uL wash buffer to the purification column
    5. Centrifuge for 60 seconds and discard flow through
    6. Centrifuge again for 60 seconds (dry spin) and discard flow through
    7. Transfer purification column to 1.5 uL microcentrifuge tube
    8. Add 30 uL elution buffer
    9. Wait 5 minutes
    10. Centrifuge for 1 minutes
    11. Store at -20 degrees C

    NUTRIENT MIX PROTOCOL

    PROTOCOL (FOR BOTH NITRITE, NITRATE) IN TECHNICAL TRIPLE KITS

    1. Autoclave 250ml flask (12)
      1. 6 for different concentrations of nitrite (0ppm,60ppm, 120ppm, 180ppm, 240ppm, 300ppm)
      2. 6 for different concentrations for nitrate (0ppm,60ppm, 120ppm, 180ppm, 240ppm, 300ppm)
    2. Add 100ul of pre-grown cells into 1 liquid culture tube
      1. Add 100ml of plain lb into a 250 flask
      2. Add 5ml of the cell stock
        1. Add 60ml of diH2O with a cell colony
      3. Grow the flask for 24 hours in the shaking incubator
    3. Add 100ml of LB into 6 unlabeled flasks and add 10ul from liquid culture tube
    4. Grow flasks for 12 hours in shaking incubator
    5. After 12 hours, transfer 50ml to each of the sterile, unused flasks
    6. Label each flask and 37 cuvettes *36 for the triplicates*
      1. Take 1ml from each flask and add it to the corresponding cuvette
      2. 36 for cells and 1 for plain lb as a negative control (blank)
    7. Measure OD600 in triplicates
      1. Take measurements 3 times for each cuvette then average data
    8. After measurement, add X grams of nitrite and nitrate to each flask
    9. Grow flasks for 4 hours in shaking incubator
    10. Take measurement again (repeat step 7)
    11. Grow flasks for 12-16 hours in shaking incubator
    12. Repeat step 7
    13. Grow flasks for 12 hours in shaking incubator
    14. Repeat step 7