Team:UNebraska-Lincoln/Experiments

UNL 2018 Improving Early Detection of The Emerald Ash Borer

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EXPERIMENTS



›LB Broth Preparation

Materials

  • LB Broth powder (Lennox)
  • 250 mL media bottle
  • Nanopure water

Procedure

  1. Weigh 6.25 grams of LB Broth powder.
  2. Add LB Broth powder into a 250 mL media bottle.
  3. Pour 250 mL of nanopure water into the media bottle.
  4. Mix well.
  5. Autoclave media bottle on cycle 2 (Liquids cycle).
  6. Retrieve and let cool to room temperature before use.



›Terrific Broth Preparation

Materials

  • 500 mL media bottle
  • Tryptone powder
  • Yeast extract
  • Autoclaved glycerol
  • Nanopure water
  • Phosphate buffer

Procedure

  1. Weigh 10 grams of tryptone powder.
  2. Weigh 12 grams of yeast extract.
  3. Mix and add 450 mL of nanopure water.
  4. Add ~5 mL of autoclaved glycerol if desired.
  5. Autoclave, retrieve, and let solution completely cool.
  6. Add 50 mL of phosphate buffer. NOTE: Phosphate buffer is prepared by adding 4.65g monopotassium phosphate and 25g dipotassium phosphate into 200 mL PCR-grade water.



›Fresh Agar Plate Preparation

Materials

  • ~ 24 petri dishes
  • 500 mL media bottle
  • Corresponding amount of 100x antibiotic stock solutions (500 µL)

Procedure

  1. Obtain and weigh 25g LB Broth powder/L of nanopure water.
  2. Obtain and weigh 15g agar powder/L of nanopure water.
  3. Mix into respective volume of nanopure water.
  4. Autoclave solution for 1 hour (the first setting).
  5. Retrieve solution and wait to cool.
  6. Add any necessary antibiotics, typically chloramphenicol (Cm) or ampicillin (Amp) stock solution (1000x) with 1-to-1000 dilution.
  7. Evenly pour solution into properly labeled petri dishes.
  8. Allow solution to then solidify.
  9. Keep agar plates in 4 °C refrigerator.



›Plasmid Mini-Prep

Materials

  • 1.5 mL eppendorf tubes
  • Thermo Scientific Plasmid Miniprep Kit
  • P1, P2, and P3 buffers
  • Endo-wash buffer
  • Zyppy-wash buffer
  • PCR-grade water

Procedure

  1. Centrifuge cell culture for 10 min at 4°C at 5,000rpm.
  2. Pour out the LB Broth Supernatant into bleach solution.
  3. Add 200 μL P1 buffer and resuspend pellet.
  4. Transfer into a fresh 1.5 mL eppendorf tube.
  5. Add 200 μL P2 buffer, mix by slowly inverting until transparent
  6. Get P3 buffer from fridge and add 400 μL. Invert tube 2-4 times and incubate for 1 minute.
  7. Centrifuge tube at max speed for 5 minutes.
  8. Transfer all the supernatant into the zymo-spin column immediately after.
  9. Place the column into a collection tube and centrifuge at 16,000 g for 30 seconds. Discard the flow-through and place the column back into the same collection tube.
  10. Add 200 µL of Endo-wash buffer to the column. Centrifuge at 16,000 g for 30 seconds.
  11. Add 400 µL of Zyppy-wash buffer to the column. Centrifuge at 16,000 g for 30 seconds.
  12. Centrifuge for two minutes at 21,000 g.
  13. Transfer the column into a clean 1.5 mL eppendorf tube, then add 30 µL of PCR-grade water directly to the column matrix.
  14. Let it stand for one minute at room temperature.
  15. Centrifuge at 16,000 g for 1.5 minutes to elute the DNA.
  16. Typically take Nanodrop measurement right after.



›Polymerase Chain Reaction

Materials

  • PCR tubes
  • dNTPs
  • 10x PCR buffer
  • MgSO4
  • Template DNA
  • Primers
  • KOD Polymerase
  • PCR-grade water
  • Thermocycler

Procedure

  1. For just one 25 µL PCR mix, the following table was applied. The reaction size was typically scaled up to meet specific experiment demand.
    Materials Volume (µL)
    dNTPs (10mM) 2.5
    10x PCR buffer 2.5
    MgSO4 1.5
    Template (~10ng/µL) 1
    Primers (10µM) 0.75 each
    KOD Polymerase 0.5
    PCR-grade water 16.25
  2. Place the prepared reaction tubes into the thermocycler and run program tailored to the target product size. Our target product size was always between 1000-3000 bp.
    Step Target Size Target Size Target Size Target Size
    - < 500 bp 500-1000 bp 1000-3000 bp > 3000 bp
    Polymerase Activation 95°C for 2 min 95°C for 2 min 95°C for 2 min 95°C for 2 min
    Denaturing 95°C for 20s 95°C for 20s 95°C for 20s 95°C for 20s
    Annealing Lowest Primer Tm for 10s (~65°C) Lowest Primer Tm for 10s (~65°C) Lowest Primer Tm for 10s (~65°C) Lowest Primer Tm for 10s (~65°C)
    Extension 70°C for 10s/kb 70°C for 15s/kb 70°C for 20s/kb 70°C for 25s/kb
    Halt 4°C for infinity 4°C for infinity 4°C for infinity 4°C for infinity



›Standard 1% Gel Electrophoresis

Materials

  • Agarose powder
  • TAE buffer
  • Erlenmeyer flask
  • Ethidium Bromide
  • Casting tray
  • Sub-Cell apparatus
  • 2-log 10kb ladder
  • 6x Loading Dye
  • DNA Product

Procedure

  1. Weigh 0.5 grams of agarose powder.
  2. Measure 50 mL of TAE Buffer into an Erlenmeyer flask and add in the agarose powder. Mix well
  3. Cover the Erlenmeyer flask and microwave for two minutes at power level ten.
  4. Let the solution cool for ~10 minutes.
  5. Add 4 µL of ethidium bromide and mix well.
  6. Pour the solution into a casting tray and let it cool for ~30 minutes.
  7. Set up the Sub-Cell apparatus and fill with enough TAE buffer to cover the gel.
  8. Carefully remove the comb.
  9. Load ~5 µL of the ladder into the leftmost well.
  10. Mix the loading dye with the DNA sample to the proper concentration and then load into well(s).
  11. Once your samples are loaded place the lid on the apparatus and run the gel at 80 volts for 1 hour.
  12. Carefully remove gel to image.



›Gel DNA Recovery

Materials

  • Zymoclean™ Gel DNA Recovery Kit
  • ADB Buffer
  • Wash Buffer
  • Hot water bath
  • Eppendorf tubes
  • PCR-grade water

Procedure

  1. Weigh the piece of cut gel, then add 3 volumes of ADB buffer to each volume of gel. (max volume in tube = 750 µL)
  2. Perform short centrifuge
  3. Incubate the 1.5 mL eppendorf tube in a 55°C water bath for 5-10 minutes.
  4. Use vortex to mix well.
  5. Add the melted agarose solution into a Zymo-spin column and place into a collection tube.
  6. Centrifuge for 30 seconds at 16,000 g. Empty the collection tube whenever necessary.
  7. Add 200 µL of Wash buffer to the column and centrifuge for 30 seconds.
  8. Repeat step 7.
  9. Centrifuge at max speed for 2 minutes.
  10. Place Zymo-Spin column into a new 1.5 mL tube.
  11. Add 8-12 µL of PCR-grade water directly to the column matrix.
  12. Centrifuge at 16,000 g for 1.5 minutes to elute the DNA.
  13. Typically take Nanodrop measurement right after.



›Sequence and Ligation Independent (SLIC) Cloning

Materials

  • Insert
  • Vector Backbone
  • 10x Buffer 2.1 NEB
  • T4 DNA Polymerase
  • PCR-grade water
  • Thermocycler
  • PCR tubes
  • Pipets

Procedure

  1. A table is offered below for guidance. 150ng of vector backbone required. Calculate the volume of vector needed for reaction.
  2. Calculate the amount of insert needed for reaction based on the vector (1:2 molar ratio).
  3. Calculate the amount of PCR-grade water needed to bring the final volume to 20 µL and load into a PCR tube.
  4. Load 2 µL of 10x Buffer 2.1 into the PCR tube.
  5. Load the vector and insert into the PCR tube.
  6. Dilute 2 µL of T4 DNA Polymerase into 10 µL of 10x Buffer 2.1.
  7. Load 1 µL of the diluted T4 DNA Polymerase into the PCR tube.
  8. Place PCR tube into the thermocycler and run SLIC Protocol (23 °C for 30 min; 75 °C for 15 min; 37 °C for 30 min; hold at 4 °C)
  9. Transform product.
  • For just one 25 µL PCR mix, the following table was applied. The reaction size was typically scaled up to meet specific experiment demand.
    Materials Volume
    Vector ~150ng
    Insert 1:2 vector to insert molar ratio
    10x Buffer 2.1 (NEB) 2 µL
    T4 DNA Polymerase (1:6 dilution in 2.1 buffer) 1 µL
    PCR-grade water Amount necessary for 20 µL reaction



    • ›Transformation

    Materials

    • Genehog Chemically Competent Cells
    • Hot water bath
    • Eppendorf tubes
    • Agar plates with antibiotic
    • Incubator (shaker)
    • Sterilized beads

    Procedure

    1. Thaw 40 µL of competent cells on ice for 5-10 minutes.
    2. Add about 100 ng of DNA then continue icing for 20-30 minutes. The added volume should be less than 10% of total volume.
    3. Place cells into 42°C water-bath for 30-60 seconds.
    4. Put cells back on ice for 2 minutes. Sterilize work area and pipet in this time.
    5. Add 1 mL of LB broth to the mixture.
    6. Leave the cells at 37°C for one hour in shaker. Meantime, prewarm agar plates.
    7. Plate cells using beads (volume varies slightly).
    8. Incubate the plate overnight at 37°C.
    9. Look for colonies.



    ›Restriction Enzyme Digest

    Materials

    • Restriction enzymes (variable)
    • 10x Cutsmart buffer
    • DNA insert/plasmid

    Procedure

    1. Prepare 14 µL of uncut DNA insert or plasmid in PCR tube.
    2. Add 2 µL of Cutsmart buffer.
    3. Add 2 µL of each desired restriction enzyme (i.e. XbaI and SpeI).
    4. Gently mix together. Total reaction volume is 20 µL.
    5. Place in incubator for 1½ hours at 37°C.
    6. Run gel electrophoresis and perform gel DNA recovery on product.
    7. Store in 4°C chamber.



    ›Subcloning (pSB1C3 vector backbone)

    Materials

    • T4 Ligase
    • 10x Ligase buffer
    • HF restriction enzymes
    • PCR tubes
    • PCR-grade water

    Procedure

    1. Perform restriction enzyme digest on vector using EcoRI and PstI.
    2. Perform restriction enzyme digest on DNA insert using EcoRI and PstI.
    3. Recover DNA after gel electrophoresis.
    4. Calculate the amount of cut vector and insert needed to have a 1:3 molar ratio. The total volume of reaction should have a DNA concentration near 10 ng/µL.
    5. Load the cut vector and insert into PCR tube.
    6. Add enough 10x Ligase Buffer so that the buffer is one-tenth the total reaction volume.
    7. Add 0.5 µL of T4 DNA Ligase into the PCR tube.
    8. Prepare a negative control by replacing the cut DNA insert portion with water.
    9. Place PCR tubes into thermocycler and run the ligation protocol.
    10. Transform product.








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