Team:DUT China A/Protocols

Protocols

Protocols

Cell Culture Binding Buffer Cell - Aptamer Circular Template DNA PAGE DNA Hydrogel

Cell Culture

  1. Aspirate off the cell medium.
  2. Wash the cells with 2 mL PBS and the aspirate off the PBS.
  3. Add 2 mL of Trypsin and wait for 3 to 5 min.
  4. Add new cell medium to the culture flask. The FBS quenches the trypsinization.
  5. Pipette medium up and down, and squirt it around the surface of the dish to remove adherent cells from the surface. Move the cells into 15 mL Falcon conical and centrifuge at 750 rpm for 5 min and discard supernatant.
  6. Add 3 mL PBS, pipette PBS up and down to mix the solution evenly. Centrifuge at 750 rpm for 5 min, discard supernatant.
  7. Add the medium according to the need and pipette the media up and down to mix the solution.
  8. Add 3 mL DMEM and move 1 mL of cells into new dishes. Spread the cells evenly by rocking the dish back and forth.
    9. DO NOT spread the cells by swirling the media in a circular motion – this results in clumping of cells in the middle of the dish.

Binding Buffer

The binding buffer is PBS buffer (pH=7.5) containing 0.1 mg/ml yeast tRNA.

Cell and Aptamer Binding

  1. Aspirate off the cell media.
  2. Wash the cells with appropriate volume of binding buffer.
  3. Incubate with appropriate amount of binding buffer for 30 min at 37 °C. (Add different volumes of binding buffer to cells cultured on different plates. For example, add 200 μL per well to a 96-well plate and 500 μL to a 48-well plate.)
  4. Aspirate the binding buffer and incubate with 200 μL of the aptamer (diluted to 200 nM with binding buffer) for 2 hours.
  5. Aspirate the solution and wash three times with PBS.

Prepare the Circular Template

  1. Heat the 5’ prime-phosphorylated linear ssDNA (30μM，8.00μL) and primer 3 (90μM，10.00μL) at 85℃ for 5 min, cool down to the room temperature (RT).
  2. Add 2 μL T4 DNA ligase (20 U/μL) and 2.22 μL 10×Ligation buffer. Incubate the mixture at 16℃ for 16 h.
  3. Add 5.00 μL Exonuclease I (5 U/μL) and 1.5 μL Exonuclease III (100 U/μL) and incubate at 37 ℃ for 4 h to digest uncycled oligonucleotides.
  4. Deactivated the enzymes by heating the mixture at 70℃ for 5 min.

DNA Polyacrylamide Gel Electrophoresis (PAGE)

  1. Prepare a 12% denaturing gel (take 20 mL as an example)
    • Add 12 mL of 20% acrylamide, 4 mL of 10×TBE and 4 mL ddH2O to the breaker.
    • Add 10% ammonium persulfate and 10 μL TEMED.
    • Mix the solution.
    • Soak the glass plates in 20% alcohol, dry them and assemble two pieces together.
    • Add the solution between two glasses by using a pipette with no air bubbles.
    • Insert the comb and place it at room temperature for 1 h to form gel.
  2. Electrophoresis
    • Install the gel into the electrophoresis tank, pour 0.5×TBE as the electrophoresis buffer and make sure the buffer is enough to cover the sample lane.
    • Add samples using a pipette (9 μL DNA sample with 1 μL of loading buffer for a total of 10 μL). Run the gel at 150 V for 10 MIN and at 80 V for another 1.5 h.
  3. Dye
    • Prepare a staining solution: 30 μL of GelRed, 10 mL of 1 M NaCl, and 90 mL of ddH2O
    • Gently take off the gel from the plate carefully and place it in a staining solution.
    • Place it in the dark for 30 min.

Preparation of DNA Hydrogel Without Cells

  1. 10 μL of aptamer-linker was placed in a PCR tube, anneal at 95 ° C for 5 min, and cooled to 20 ° C.
  2. Add 30 ul of TE buffer (pH = 7.5) and10 μL of circular template to the tube, mix and incubate in a 30 ° C incubator for 2 h.
  3. Add 8 μL of 10X phi29 reaction buffer (pH=7.4), 0.4 μL of BSA, 20 μL dNTP mixer, 1 ul phi29 DNA polymerase (Haigene) to the tube, and incubate in a 30 ° C incubator for 40 min.
  4. Add 5ul of primer 2 and primer 3, incubate at 30 ° C.

Preparation of DNA hydrogel with cells

  1. Add 30 ul of TE buffer (pH = 7.5) to the cells that have bound with aptamer, 10 μL of the circular template, and mix and incubate in a 37 ° carbon dioxide incubator for 2 h.
  2. Add 8 μL of 10X phi29 reaction buffer (pH=7.4), 0.4 μL of BSA, 20 μL dNTP mixer, 1 ul phi29 DNA polymerase (Haigene) to the culture dish, and incubate for 40 min in a 37 ° C carbon dioxide incubator.
  3. Add 5 ul of primer 2 and primer 3 and incubate in a 37 ° carbon dioxide incubator.