Team:McMasterU/DNAzyme

Protocols


Testing Fluorescence of Bacterial Plates

Date: May 18
Reagents:

  • 3x DNAzyme stock: 13.35 uL (200nM final concentration)
  • 2x selection buffer (150uL)
  • dH2O: 136.65uL


    • Protocol:

    1. The DNAzyme stock solution in the previous slide was created in an Eppendorf tube.
    2. 100 uL of DNAzyme stock solution was pipetted into Plates 1,2, and 3. A hockey stick was used to spread the solution over the surface.
    3. Plates 1-3 were left for 5 minutes to dry (with plate lids on).
    4. A disposable inoculation loop was used to streak nothing onto Plates 1 and 5.
    5. Disposable inoculation loops were used to streak E. coli on Plates 2 and 4 and B. subtilis on Plate 3.
    6. Plates were placed upside down in an incubator at 37℃.

    Plates Segmentation:

  • only DNAzyme
  • E. coli with DNAzyme
  • B. subtilis with DNAzyme
  • E. coli only
  • Nothing



    • Fluorophore Concentrations

    Date: May 23
    Reagents:

  • FDNA
  • dH2O
  • Hockey stick
  • LB agar plates
  • UV box


    • Protocol:

    1. 7 different concentrations of fluorescent DNA with the fluorophore were created. These concentrations were 0, 100, 250, 500, 1000, 2500, and 5000 nM.
    2. 10 uL from each of the concentrations were dropped into the middle of a quadrant on a plate.
    3. A hockey stick was used on each quadrant to spread the fluorescent DNA.
        *Did not spread because the dot of solution had set into the agar and therefore did not spread with the hockey stick. This time we dotted the plates and immediately used the hockey stick after putting each dot onto each quadrant.



    Optimizing Concentrations & Cleavage


    Date of Experiment: May 26

    Materials/Reagents:

    • 3 agar plates
    • 100uL of:
    • 2.5uM FDNA, 5.0uM FDNA
    • 2.5uM FQ substrate (RS28), 5.0uM FQ substrate (RS28)
    • Gycerol stock of E.coli


    Protocol:

    3 plates were split into quadrants with 25uL FDNA and FQ substrate stock on each quadrant: Plate 1 (2.5uM): FDNA, Negative, E. coli, FDNA + E. coli Plate 2 (2.5uM): FQ substrate, negative, FQ substrate + 0.5uM 0.2uM NaOh, FDNA Plate 3 (5.0uM): FQ substrate, negative, FQ substrate with 0.5uM 0.2M NaOH, FDNA E. coli was streaked onto its designated quadrants on plate 1 Plate 2 and 3 were checked under blue light for fluorescence after NaOH applied for 15 minutes All plates were placed in 37C incubator overnight Plates were visualized using the Typhoon. The following scans were performed using the blue laser (488nm): 526 SP Fluorescein, Cy2, AlexaFlour488 Emission Filter 520 BP CY2, ECL+, Blue FAM Emission Filter Emission Filter