Team:NIPER-Guwahati/notebook

Calendar

May 2017

  • On 16th, Our idea was born :)

  • We planned out our strategy on how we could use DNAzymes in the fight against cancer.

  • 17th May: Titrated mRNA against streptavidin beads (DynaBeads)

  • 18th May: Standardised the optimal concentration of mRNA that can be bound to the beads.

June 2017 - July 2017

  • 2nd June: Standardisation of DNAzyme reaction

  • 11th June to 27th July: Performed 10 rounds to obtain DNAzymes capable of cleavage reaction Evolution and selection of DNAzymes and biotin approach

  • 21st June to 21st July: Preparation of calcium competent DH5α cells.

August 2017

  • 25th July to 11th August:

    • Synthesised cDNA from mRNA of various cancer cell lines.
    • Attempted PCR amplification of BCl-2 coding sequence from the cDNA… We were not successful :(

  • 24th August to 5th September: Used NEB phage display kit to select RNA specific to HL-60 leukaemia cancer cells

  • 25th August onwards: DNAzymes were cloned using TA cloning vector

September 2017

  • 5th September: Confirmed successful cloning of DNAzymes

  • 5th September to 3rd October: Used NEB phage display kit to select cell penetrating peptides specific to A375 melanoma cancer cells

  • 25th September: Submitted DNAzymes for sequencing

October 2017

  • 12th October to 22nd October:

    • Cloned pSB1C3
    • Isolation of pSB1C3 plasmid

    • 23rd October:

      • Confirmed cleavage activity of DNAzymes using RT-PCR
      • Transfection of A375 cancer cell lines
      • RNA isolation and cDNA synthesis from isolated DNAzyme mRNA

    • 26th October: Ligated DNAzyme insert into pSB1C3 vector
      27th October: Focusing on Wiki page and uploading information regarding catalytic activity of DNAzymes




Protocols

Preparation of DNAzymes

Preparation of Beads

  1. Take two 1.5 mL centrifuge tubes. Take 50 µL of magna beads in each.
  2. Prewash the beads with 100 µL of solution A twice and soup out the liquid.
  3. Prewash the beads with 100 µL of solution B twice and soup out the liquid clearly.
  4. Take 1 µL of biotinylated BCl-2 mRNA from 100 µM stock solution and add into 100 µL of 2X binding and washing buffer.
  5. Incubate the mRNA (100 µL) into beads at 37°C for 15 min at 150 rpm shaking condition.
  6. Soup out unbound mRNA.
  7. Wash the unbound mRNA twice with 1X wash buffer.

Preparation of DNA Library

  1. Take 1 µL of DNA library from 100 µM stock solution and dilute into 100 µL of binding buffer.
  2. Heat the library at 95ᵒC for 10 minutes and snap chill it.
  3. Then incubate snap chilled library with beads at 37ᵒC for 10 minutes with 150 rpm in incubator shaker.
  4. After 10 minutes soup out the unbound library.
  5. Wash the beads 8 times with wash buffer.
  6. Add 100 µL of cleavage buffer (150mM KCl, 2 mM MgCl2)
  7. Incubate at 37°C for 3 hrs with 150 rpm shaking condition.
  8. Soup out the cleaved molecules.
  9. Set up the following PCR reaction in 3 tubes.
    10. Contents Quantity
    Master Mix 25 µL
    Cleaved molecules 24.5µL
    (100μM) Forward primer library 0.25µL
    (100μM) Reverse primer library 0.25µL
    Total volume per tube 50µL

    So, the master reaction will be,
    Contents Quantity
    Master Mix 75 µL
    Cleaved molecules 73.5µL
    (100μM) Forward primer library 0.75µL
    (100μM) Reverse primer library 0.75µL
    Total volume 150µL
  10. Run the 5 µL of PCR product in 1.5 % gel using urea-PAGE electrophoresis.
    11. Control Reaction: Add 100μL wash buffer to beads and incubate for 3 hrs at 37°C and 150 rpm.

    Urea-PAGE Gel Electrophoresis

    We ordered readymade urea page gel and loaded the sample with TBE Urea sample buffer.
    Crush and soak method.
    Single or double stranded DNA molecules once separated, were extracted from the urea PAGE gel by crush and soak method. The principle involved was separation of DNA molecules by simple diffusion method. Gel piece of interest was cut down from the gel, dried and the gel was made into fine particles. 3 to 4 volume of the 1mM EDTA +0.5M ammonium acetate solution was added to the dried and fine powdered urea gel and incubated for a period of 6 to 8 hours at 55°C.After the incubation time the sample was centrifuged at 12000 rpm for 5 minutes and collected the supernatant that contains DNA.

    Transfection Of DNAzymes

    1. Heat DNAzyme samples to 95°C to denature and make it single stranded.
    2. In eppie tubes, add 10μL each of DNAzyme to 175μL of optiMEM.
    3. Also prepare a control consisting of 10μL milliQ water and 175μL optiMEM.
    4. Prepare oligofectamine mix.
      • In a 6 well culture plate, add 6μL of oligofectamine and 45μLof optiMEM to each well and pipette it 15 times.
      • Incubate at room temperature for 8 minutes.
    5. Combine DNAzyme mix with oligofectamine mix (15μL/well)
    6. Incubate for 20 min at room temperature.
    7. Remove growth media and wash once with serum free media.
    8. Replenish with 800μL of serum free media.
    9. Add 200μL of complexes drop by drop.
    10. Incubate at 37°C for 4hrs.
    11. Prepare recovery media: 3mL FBS +7mL media.
    12. After 4hrs, add 500μL of recovery media to each well.

    Isolation Of RNA Using Trizol Reagent

    1. Harvesting of cells
      1. Rinse cells with cold PBS once.
      2. Scrape the cells with cell scraper and PBS.
      3. Centrifuge the cell suspension at 2000 rpm for 5 minutes.
      4. Remove PBS and take cell pellet.
    2. Cell lysis
      1. Add 1mL of Trizol for cell pellet of 5 to 6×106 cells. Vortex thoroughly and pipette several times.
      2. Incubate homogenized sample for 5 minutes at room temperature to permit the complete dissociation of nucleoprotein complex.
    3. Phase separation
      1. Add 0.2mL of chloroform per mL of Trizol reagent.
      2. Vortex sample vigorously for 15 sec and incubate at room temperature for 2-3 minutes.
      3. Centrifuge sample at 12,000xg for 15 minutes at 2-8°C.
      4. Three phases will form – lower red phenol-chloroform phase, an interphase and an upper aqueous phase which contains RNA.
      5. Take the aqueous phase out carefully without disturbing.
    4. RNA precipitation
      1. Add 0.5mL isopropyl alcohol for 1mL of Trizol.
      2. Centrifuge at 12,000xg for 10 minutes at 2-4°C.
    5. RNA Wash
      1. Add 1mL 75% ethanol for 1mL Trizol.
      2. Centrifuge at 7,500xg for 5 minutes at 2-8°C
      3. Remove ethanol.
      4. Airdry RNA pellets for 5- 10 minutes.
      5. Re-suspend RNA with DEPC treated water.