Team:CCU Taiwan/Protocol







Preparation procedure for the surface modification of iron oxide nanoparticles with nanoprobe

  1. Take 5 μL / mL of carboxylated beads (COOH group) into 1.5 mL centrifuge tube and then place the magnet externally for about 1 minute and remove the supernatant layer.
  2. Then, dissolve the pallet with MES buffer (50 mM MES + 0.15M NaCl, pH 6.2) by adding 1000 μL volume place the magnet for about 1 minute and then remove the supernatant layer.
  3. Add a 1:1 mixture of 50 μL 0.01M EDC and 0.017M NHS|MES buffer and shake at room temperature for 1 hour.
  4. Apply magnet externally about 1 minute again and then remove the supernatant. Then, add 1000 μL MES buffer to dissolve it.
  5. Then add 5 µL of 5 x 10-6 g/mL nanoprobe to 950 µL and shake at room temperature for 30 minutes.
  6. After removing the supernatant, add 500 µL Ethanolamine pH 8.5 and shake at room temperature for 30 minutes and remove the supernatant.
  7. Finally, add PBS buffer back and store it in 1x PBS, pH 8.4 and keep in refrigerator at 4°C.



Preparation of chemicals

  1. EDC (molar weight: 191.7): 0.01M - 0.0019 g to 1mL distilled water
  2. NHS (molar weight: 115.09): 0.017M - 0.0020 g to 1mL distilled water
  3. MES buffer (195.24 g/mole) + NaCl (58.44 g/mole) - 50 mM MES + 0.15 M NaCl at pH 6.2 - 0.4881 g (MES) + 0.4383 g (NaCl) to 50 mL distilled H2O.

    4. • Mass (g) = conc. (mol/L) x volume (L) x formulated wight (g/mol)



Gel extraction

  1. After running the DNA gel in TAE buffer, cut out the desired DNA band ( ≤ 350 mg) with a clean scalpel. Weight the gel slice in a microcentrifuge tube.
  2. Add equal volume of Binding Buffer.
  3. Incubate at 60℃ for 10 minutes and vortex the mixture, or until the gel slice is completely dissolved.
  4. Insert the Spin column into a collection tube, transfer the solution into the spin column and spin at full speed for 1 minute, and discard the flow-through liquid in the collection tube.
  5. Add 600 µL of Washing Buffer and centrifuge at full speed for 1 minute. Repeat this washing procedure for one more time.
  6. Discard the flow-through liquid and centrifuge for 3 minutes at full speed to remove residual trace of ethanol.
  7. Transfer the spin column into a new microcentrifuge tube and add 50-100 µL of elution Buffer or H2O into the column and wait for 1-2 minutes.
  8. Centrifuge at full speed for 1 minute to elute the DNA. Store the DNA in the microcentrifuge tube at -20℃.



Plasmid purification

  1. Centrifuge 1-3 mL bacterial culture at 12,000 g for 1 minute in the centrifuge tube. Discard the supernatant and remove excess media.
  2. Resuspend the pellet in 200 µL of Solution I, and mix well by pipetting or vortex.
  3. Add 200 µL of Solution II, mix gently by inverting the tube 4-6 times, do not vortex.
  4. Add 200 µL of Solution III, mix gently by inverting the tube 4-6 times, do not vortex.
  5. Centrifuge the lysate for 10 minutes at 12000 g speed in a microcentrifuge.
  6. Insert one spin column into one collection tube, transfer supernatant from step 5 into the spin column, centrifuge at 12000 g speed in a microcentrifuge for 1 minute.
  7. Discard the flow-through liquid, add 600 µL of Wash Buffer into the spin column, centrifuge at 12000 g speed for 1 minute. Repeat this wash procedure one more time.
  8. Centrifuge at 12000 g speed for 3 minutes to remove residual ethanol.
  9. Transfer the spin column to a new microcentriguge tube, add 50-100 µL of Elution Buffer or H2O into spin column, and incubate at room temperature for 1 minute.
  10. Centrifuge at 12000 g speed for 1 minute to elute plasmid, and store the DNA at -20℃



In Vitro Transcription

  1. Precipitate the DNA by adding 1/10th volume of 3 M Sodium Acetate Solution (pH 5.2), and two volumes of ethanol. Incubate at -20°C for at least 30 minutes and collect the pellet by centrifugation.
  2. Remove the supernatant and rinse the pellet with 500 µL of 70% ethanol.
  3. Resuspend the pellet in DEPC-treated water.
  4. Combine the following reaction components at room temperature in the order given:
    Substance Volume (unit)
    DEPC-treated water to 20 µL
    5X TranscriptAid Reaction Buffer 4 µL
    ATP/CTP/GTP/UTP mix 8 µL
    Template DNA 1 µg
    TranscriptAid Enzyme Mix 4 µL
    Total volume 20 µL
  5. Mix thoroughly, spin briefly to collect all drops and incubate at 37°C for 2 hours.
  6. 20 µL reaction mixture add 115 µL of DEPC-treated water and 15 µL of 3 M Sodium Acetate Solution (pH 5.2). Mix thoroughly.
  7. Extract with an equal volume of 1:1 phenol (pH 4.7)/chloroform mixture, and then twice with equal volume of chloroform. Collect the aqueous phase and transfer to a new tube.
  8. Precipitate the RNA by adding 2 volumes of ethanol. Incubate at –20°C for at least 30 minutes and collect the pellet by centrifugation.
  9. Remove the supernatant and rinse the pellet with 500 µL of cold 70% ethanol. Resuspend the RNA in 20 µL of DEPC-treated water.
  10. Store the RNA at -80°C.



Produce ssDNA from negative control plasmid

  1. ScaI (restriction enzyme) digestion of negative control plasmid.
  2. Denature restriction enzyme.
  3. Clean up the solution.
  4. Denature linear negative control DNA at 95℃ for 30 minutes.
  5. Cool down the solution immediately at -20℃ for 20 minutes.
    6. Substance Volume (μL)
    ScaI (restriction enzyme) 1
    cutsmart (10X buffer) 6
    10 μg Negative control plasmid (642.8 ng/μL) 15
    ddH2O 38
    Total volume 60
  6. Gel extraction for specific ssDNA



Broth culture and protein expression

  1. E. coli DH5α transformed with plasmid encoded with Cas12a protein (pHMT-Cas12a) was cultured in 5 mL Terrific Broth (TB) at 37℃ overnight with shaking.
  2. Freshly expand BL21 with pHMTCas12a vector by adding 500 μL bacterial suspension from TB+AMP medium cultured last night to 50 mL fresh TB medium. The fresh TB medium was cultured at 37℃ with shaking.
  3. Grow the BL21 with pHMT-Cas12a vector to exponential phase (OD 0.6~0.8) at 37℃ with shaking.
  4. Induction of Cas12a protein expression in BL21 by adding 50 μL of 0.2 mM IPTG in 50 mL TB culture and culture at 16 ℃ for 14 hours.



Protein purification by Ni2+-magnetic beads

  1. Harvest BL21 by centrifuging at 8000 r.p.m., 5 minutes, discard the medium.
  2. Add 1 mL Cas12a lysis buffer to resuspend cell pellet into each eppendorf.
  3. Sonication carried out on ice as condition shown in table:

    4. ★cycle the step for 3 minutes,strength (Watts RMS) for 5
    break BL21 pause
    time(sec) 10 10
  4. Collected soluble fraction after sonication by centrifuging at 4℃ for 15 minutes at 13200 r.p.m.
  5. Wash the Ni2+-magnetic beads three times (1000 μL lysis buffer/one time).
  6. Incubate soluble fraction with Ni2+-magnetic beads for 1 hour.
  7. Wash the Ni2+-magnetic beads three times (1000 μL TEV buffer/one time).
  8. Elute Ni2+-magnetic beads captured Cas12a by treating TEV enzyme.

    9. ★lysis buffer
    Substance Volume (unit)
    50 mM Tris-HCl, pH7.5 7.882 g
    500 mM NaCl 29.22 g
    5%(v/v)glycerol 50 mL
    20 mM ß-ME 1.399 mL from 14.3 M
    0.5 mM PMSF 9.671 mL from 14.3 M
    mQH2O add to 1000 mL
    total volume 1000 mL
    • (v/v) which means after mQH2O is added to 950 mL, 5%*1000 = 50 mL glycerol is added at last. BME, PMSF, should be added in fresh.

    ★TEV buffer
    Substance Volume (unit)
    0.5 M Tris-HCl 3.94 g
    0.005 M EDTA 0.09 g
    0.2 M ß-ME 0.7 mL from 14.3 M
    mQH2O add to 50 mL
    total volume 50 mL



Protein purification by Ni2+ chelating sepharose column

  1. Get BL21 cell from -80℃.
  2. Smash Cas12a induced BL21 cells by French press with pressure 1500 psi .
  3. Collected soluble fraction by centrifuging smashed cells at 17500 rcf, 30 minutes at 4℃ .
  4. Capture Cas12a protein by incubating soluble fraction with Ni2+ -magnetics beads for 30 minutes at RT with shaking.
  5. Washed Ni2+ -magnetics beads with lysis buffer, detect the flow though by FPLC.
  6. Eluted by elution buffer (with imidazole).

    7. ★Buffer used for FPLC
    Buffer Name Volume
    buffer A lysis buffer 350 ml
    buffer B elution buffer: 300 mM imidazole in lysis buffer 250 ml



Cas12a protein trans cleavage

  1. Combine Cas12a protein with crRNA at 37℃ for 10 minutes.
  2. Add linear positive control DNA and linear negative control DNA for cleavage at 37℃ for 50 minutes.
  3. Denature Cas12a protein at 65℃ for 10 minutes.

    4. ★Sample for positive control
    Substance Volume (μL)
    36 nM crRNA 0.5
    30 nM LbCas12a 1
    2.1 buffer 3
    ddH2O 24.9
    9 nM linear positive control DNA (638.5 ng/μL) 0.6
    Total volume 30

    ★Sample for negative control
    Substance Volume (μL)
    36 nM crRNA 0.5
    30 nM LbCas12a 1
    2.1 buffer 3
    ddH2O 24.66
    9 nM linear negative control DNA (570.8 ng/μL) 0.84
    Total volume 30



Cas12a protein cis cleavage

  1. Combine commercial Cas12a protein with commercial crRNA at 37℃ for 10 minutes.
  2. Add linear positive control DNA and ssDNA for cleavage at 37℃ for 50 minutes.
  3. Denature Cas12a protein at 65℃ for 10 minutes.

    4. ★Sample for Cas12a protein trans cleavage
    1. positive control
      2. Substance Volume (μL)
      9 nM linear positive control DNA (638.5 ng/μL) 0.6
      ddH2O 26.4
      2.1 buffer 3
      Total volume 30
    2. ssDNA
      3. Substance Volume (μL)
      6.25 μM commercial ssDNA (57bp) 1.87
      ddH2O 25.13
      2.1 buffer 3
      Total volume 30
    3. Cas12a protein trans cleavage
      4. Substance Volume (μL)
      36 nM crRNA 0.5
      30 nM LbCas12a 1
      2.1 buffer 3
      ddH2O 23.03
      9 nM linear positive control DNA (638.5 ng/μL) 0.6
      3.1 μM commercial ssDNA (57bp) 1.87
      Total volume 30