Team:Hong Kong UCCKE/Protocols

Restriction Enzyme Digestion
Materials needed
  • 70% ethanol
  • Paper towels
  • Ice
  • Container for ice
  • Lab marker/Sharpie
  • pSB1C3 linearized plasmid backbone (25ng/μl) (kit)
  • Part (25ng/μl)
  • 0.6ml tubes (kit)
  • 10X Red Buffer
  • NEB enzymes: EcoRI, PstI
  • Thermocycler, or waterbath and thermometer

Protocols
  1. Clean the lab bench by wiping down with 70% ethanol and paper towels.
  2. Keep all enzymes and buffers used in this section on ice.
  3. Prepare a reaction mix by adding reagents in the order indicated in Table 1.
  4. For 1 insert (μl) For vector (μl)
    Water 6 11
    10X Red Buffer 2 2
    DNA 10 5
    EcoR1 1 1
    Pst1 1 1
  5. Incubate at 37°C for 20 minutes.
Gel Electrophoresis
Materials needed
  • Agarose powder
  • TAE Buffer
  • Hot plate and Water Bath
  • SYBR Green dye
  • Gel Tank
  • DNA Samples
  • Loading Dye
  • 1KB ladder marker
  • Power supply

Prepare Agarose Gel
Gel solution (1%)
  1. Measure 0.6 g Agarose powder and add it to a 200 ml conical flask
  2. Add 60 ml TAE Buffer to the flask
  3. Cover the opening with aluminium foil
  4. Melt the agarose using the hot plate until the solution becomes clear
  5. Let the solution cool to about 50-55°C using water bath, swirl the flask occasionally to cool evenly.
  6. Add 6μl of SYBR Green dye into the solution.
    *SYBR Green is light-sensitive, keep it dark*
  7. Seal the ends of the casting tray with two layers of tap
  8. Place the combs in the gel casting tray
  9. Pour the melted agarose solution into the casting tray and let cool until it is solid, it should appear milky white
  10. Carefully pull out the combs and remove the tape
  11. Place the gel in the electrophoresis chamber
  12. Add enough TAE Buffer (About 120 ml) so that there is about 2-3 mm of buffer over the Gel

Loading The Gel
  1. Record the order each sample will be loaded on the gel, including who prepared the sample, the DNA template -
    from what organism did the DNA came from, controls and ladder.
  2. Add 2μl loading buffer on parafilm. (Same number as sample)
  3. Carefully pipette 10μl of each sample and mix with loading buffer.
  4. Load the mixed sample to the corresponding well.
  5. Pipette 10μl of the DNA ladder marker into at least one well of each row on the gel.

Running The Gel
  1. Place the lid on the gel box, connecting the electrodes.
  2. Connect the electrode wires to the power supply, making sure the positive (red) and negative (black) are correctly connected.
  3. Turn on the power supply to about 120 volts, current:300
  4. Check to make sure the current is running through the buffer by looking for bubbles forming on each electrode.
  5. Check to make sure that the current is running in the correct direction by observing the movement of the blue loading dye – this will take a couple of minutes (it will run in the same direction as the DNA).
  6. Let the power run until the blue dye approaches the end of the gel.
  7. Turn off the power.
  8. Disconnect the wires from the power supply.
  9. Remove the lid of the electrophoresis chamber.
  10. Using gloves, carefully remove the tray and gel.
Gel purification
Materials
We are using QIAquick Gel Extraction Kit for Gel purification

Protocols
  1. Excise the DNA fragment from the agarose gel with a clean, sharp scalpel.
  2. Weigh the gel slice in a colorless tube. Add 3 volumes Buffer QG to 1 volume gel (100 mg gel - 100 pl). The maximum amount of gel per spin column is 400 mg. For >2% agarose gels, add 6 volumes Buffer QG.
  3. Incubate at 50°C for 10 min (or until the gel slice has completely dissolved). Vortex the tube every 2-3 min to help dissolve gel. After the gel slice has dissolved completely, check that the color of the mixture is yellow (similar to Buffer QG without dissolved agarose). If the color of the mixture is orange or violet, add 10 pl 3 M sodium acetate, pH 5.0, and mix. The mixture turns yellow.
  4. Add 1 gel volume isopropanol to the sample and mix.
  5. Place a QIAquick spin column in a provided 2 ml collection tube or into a vacuum manifold. To bind DNA, apply the sample to the QIAquick column and centrifuge for 1 min or apply vacuum to the manifold until all the samples have passed through the column. Discard flow-through and place the QIAquick column back into the same tube. For sample volumes of >800 μl, load and spin/apply vacuum again.
  6. If DNA will subsequently be used for sequencing, in vitro transcription, or microinjection, add 500 μl Buffer QG to the QIAquick column and centrifuge for 1 min or apply vacuum. Discard flow-through and place the QIAquick column back into the same tube.
  7. To wash, add 750 μl Buffer PE to QIAquick column and centrifuge for 1 min or apply vacuum. Discard flow-through and place the QIAquick column back into the same tube.
    Note: If the DNA will be used for salt-sensitive applications (e.g., sequencing, bluntended ligation), let the column stand 2–5 min after addition of Buffer PE.
  8. Centrifuge the QIAquick column in the provided 2 ml collection tube for 1 min to remove residual wash buffer.
  9. Place QIAquick column into a clean 1.5 ml microcentrifuge tube.
  10. To elute DNA, add 50 μl Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the center of the QIAquick membrane and centrifuge the column for 1 min. For increased DNA concentration, add 30 μl Buffer EB to the center of the QIAquick membrane, let the column stand for 1 min, and then centrifuge for 1 min. After the addition of Buffer EB to the QIAquick membrane, increasing the incubation time to up to 4 min can increase the yield of purified DNA.
  11. If purified DNA is to be analyzed on a gel, add 1 volume of Loading Dye to 5 volumes of purified DNA. Mix the solution by pipetting up and down before loading the gel.
Ligation
Materials needed
  • T4 DNA Ligase Reaction Buffer
  • T4 DNA Ligase
  • Microcentrifuge
  • Thermocycler, or waterbath and thermometer
  • Restriction Digest: pSB1C3 linearized plasmid backbone
  • Restriction Digest: Insert

Protocol
  1. Add 2ul from the pSB1C3 linearized plasmid backbone digest.
  2. Add 3.3μl from the Part digest.
  3. Add 1μl of T4 DNA Ligase Reaction Buffer.
  4. Add 0.5μl of T4 DNA Ligase (keep this at -20°C until use!).
  5. Mix by gently pipetting up and down 3x. Do not vortex; this inactivates the enzymes. Place tube in microcentrifuge for a quick 5 second spin or flick the tube to collect the mixture at the bottom.
  6. Incubate at 16°C for 30 minutes, then at 80°C for 20 minutes using a thermocycler.
Transformation
Materials needed
  • Competent cells
  • Ligation Products
  • Ice
  • Water bath and hot plate for heat shock
  • LB broth
  • Centrifuge
  • Agar plate

Protocol
  1. Wait until the competent cells melt in room temperature
  2. Pipette all the ligation products into the tube of cells
  3. Incubate the tube of cells in ice for about 20 minutes
  4. Heat shock at 42 ℃ for 1-2 minutes
  5. Incubate in ice for 5 minutes
  6. Add in 1mL of LB broth into the tube of cells
  7. Shake it at 37 ℃ 220rpm for approximately 45 minutes to 2 hours
  8. Centrifuge it 13000rpm for 3 mins
  9. Take 1mL sample out of the tube of cells
  10. Resuspend it and spread it on an agar plate
Miniprep
Materials needed
  • QIAprep® Spin Miniprep Kit

Protocol
  1. Pellet 1-5 ml bacterial overnight culture by centrifugation at >8000 rpm (6800 x g) for 3 min at room temperature (15-25°C).
  2. Resuspend pelleted bacterial cells in 250μl Buffer P1 and transfer to a micro centrifuge tube.(1.5ml)
  3. Add 250 μl Buffer P2 and mix thoroughly by inverting the tube 4-6 times until the solution becomes clear. Do not allow the lysis reaction to proceed for more than 5 min.
    If LyseBlue reagent is used, the solution will turn blue.
  4. Add 350 μl Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times. If LyseBlue reagent is used, the solution will turn colorless.
  5. Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge.
  6. Apply the supernatant from step 5 to the QlAprep spin column by decanting or pipetting. Centrifuge for 30-60 s and discard the flow-through
  7. Recommended: Wash the QlAprep spin column by adding 500 μl Buffer PB. • Centrifuge for 30-60 s and discard the flow-through
  8. Wash the QlAprep spin column by adding 750 pl Buffer PE. • Centrifuge for 30-60 s and discard the flow-through.
  9. Centrifuge for 1 min.
  10. Place the QlAprep column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50p1 Buffer EB (10 mM Tris•a, pH 8.5) or water to the center of the QlAprep spin column, let it stand for 1 min, and centrifuge for 1 min.
Assay_A
Materials needed
  • Plate reader SLS_F17020
  • 96-well plate
  • Distilled water
  • Different concentration of uric acid
  • Engineered cells (With BBa_K2197300)
  • Competent cell

Protocol
  1. Centrifuge 4 ml of cells and discard the LB
  2. Resuspend them with 200μl of PBS
  3. Pipette 50μl of water into A1-A6
  4. Pipette 50μl of 1x10^-4 mg/dL uric acid into B1-B6
  5. Pipette the same volume of different concentrations of uric acid into the wells (B1-G12) as the table below:
  6. 123456789101112Concentration of Uric Acid
    A0
    B1x10^-4
    C2x10^-4
    D4x10^-4
    E6x10^-4
    F8x10^-4
    G1x10^-3
    H1x10^-3
  7. Add 50 μl of cultured cells (Competent cells into Column 1-3; engineered cell with K2197300 into Column 4-6)
  8. Let it set for 30 minutes
  9. Place it in the plate reader to measure the Green Fluorescence level
Assay_B
Materials needed
  • Plate reader SLS_F17020
  • 96-well plate
  • Distilled water
  • Different concentration of uric acid
  • Engineered cells (With BBa_K2197300, BBa_K2197400, BBa_K2197502)
  • Competent cell

Protocol
  1. Grow cell with BBa_K2197300 overnight in 10 mL LB broth
  2. Prepare a flask of 40 mL LB broth with 1x10^-3 uric acid concentration (flask A)
  3. Prepare another flask of 40 mL LB broth with SOD medium (flask B)
  4. Dilute the LB broth in flask A into 8x10^-4, 6x10^-4, 4x10^-4, 2x10^-4 and 1x10^-4 uric acid concentration respectively with the LB broth in flask B with a total of 10 ml volume.
  5. Add 1 mL engineered cell grown previously into all concentrations LB broth in flask A
  6. Shake the engineered cells for an hour
  7. Centrifuge 2 mL of cells in five separate tubes respectively and discard the LB
  8. Resuspend them with 200μl of PBS in each tube
  9. Pipette 50 μl cells into the well plate as the following
    1-3: competent cells
    4-6: BBa_2197300
  10. 123456789101112Concentration of Uric Acid
    A0
    B1x10^-4
    C2x10^-4
    D4x10^-4
    E6x10^-4
    F8x10^-4
    G1x10^-3
    H1x10^-3
  11. Place it in the plate reader to measure the Green Fluorescence level
Assay_C
Materials needed
  • Plate reader SLS_F17020
  • 96-well plate
  • Distilled water
  • Different concentration of uric acid
  • Engineered cells (With BBa_K2197300, BBa_K2197400)
  • Competent cell

Protocol
  1. Centrifuge 4 ml of cells and discard the LB
  2. Resuspend them with 200ul of PBS
  3. Pipette 50μl of water into A1-A9
  4. Pipette 50μl of 1x10^-4 mg/dL uric acid into B1-B9
  5. Pipette the same volume of different concentrations of uric acid into the wells (B1-G12) as the table below:
  6. 123456789101112Concentration of Uric Acid
    A0
    B1x10^-4
    C2x10^-4
    D4x10^-4
    E6x10^-4
    F8x10^-4
    G1x10^-3
    H1x10^-3
  7. Add 50μl of cultured Cells (Competent cell into Column 1-3; BBa_K2197400 into Column 4-6) and let it set for 30 minutes
  8. Add 50μl of cultured Cells (BBa_K2197300) into Column 1-9
  9. Let it set for 30 minutes
  10. Place it in the plate reader to measure the Green Fluorescence level
Assay_D
Materials needed
  • Plate reader SLS_F17020
  • 96-well plate
  • Distilled water
  • Different concentration of uric acid
  • Engineered cells (With BBa_K2197300, BBa_K2197400, BBa_K2197502)
  • Competent cell

Protocol
  1. Centrifuge 4 ml of cells and discard the LB
  2. Resuspend them with 200ul of PBS
  3. Pipette 50μl of water into A1-A12
  4. Pipette 50μl of 1x10^-4 mg/dL uric acid into B4-B12
  5. Pipette the same volume of different concentrations of uric acid into the wells (B1-G12) as the table below:
  6. 123456789101112Concentration of Uric Acid
    A0
    B1x10^-4
    C2x10^-4
    D4x10^-4
    E6x10^-4
    F8x10^-4
    G1x10^-3
    H1x10^-3
  7. Add 50μl of cultured Cells (Competent cell into Column 1-3; BBa_K2197500 into Column 4-6) and let it set for 30 minutes
  8. Add 50μl of cultured Cells (BBa_K2197500) into Column 1-9
  9. Let it set for 30 minutes
  10. Place it in the plate reader to measure the Green Fluorescence level
Miniprep
Materials needed
  • QIAprep® Spin Miniprep Kit

Protocol
  1. Pellet 1-5 ml bacterial overnight culture by centrifugation at >8000 rpm (6800 x g) for 3 min at room temperature (15-25°C).
  2. Resuspend pelleted bacterial cells in 250μl Buffer P1 and transfer to a micro centrifuge tube.(1.5ml)
  3. Add 250 ul Buffer P2 and mix thoroughly by inverting the tube 4-6 times until the solution becomes clear. Do not allow the lysis reaction to proceed for more than 5 min.
    If LyseBlue reagent is used, the solution will turn blue.
  4. Add 350 ul Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times. If using LyseBlue reagent, the solution will turn colorless.
  5. Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge.
  6. Apply the supernatant from step 5 to the QlAprep spin column by decanting or pipetting. Centrifuge for 30-60 s and discard the flow-through
  7. Recommended: Wash the QlAprep spin column by adding 500 ul Buffer PB. • Centrifuge for 30-60 s and discard the flow-through
  8. Wash the QlAprep spin column by adding 750μl Buffer PE. • Centrifuge for 30-60 s and discard the flow-through.
  9. Centrifuge for 1 min.
  10. Place the QlAprep column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50p1 Buffer EB (10 mM Tris•a, pH 8.5) or water to the center of the QlAprep spin column, let it stand for 1 min, and centrifuge for 1 min.

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