Team:McMasterU/DNAzyme

Contents

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:


    1. 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
    2. E. coli was streaked onto its designated quadrants on plate 1
    3. Plate 2 and 3 were checked under blue light for fluorescence after NaOH applied for 15 minutes
    4. All plates were placed in 37C incubator overnight
    5. 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

    Experiment 1.a - M9 vs LB Autofluorescence via Agar Thickness


    Date of Experiment: June 12-13
    Goal: Testing different thicknesses of M9 and LB plates to find the minimal autofluorescence. Determine the optimal medium (M9 vs LB) and the optimal volume/thickness
    Different thicknesses by volume spread on a 5 mL plate: 5 mL, 3.5 mL, 2.5 mL, 1 mL


    Materials:

    • LB (12 mL) & M9 (12 mL)
    • Small plates (typically 5 mL agar capacity)
    • UV light & Typhoon Imaging
    • FDNA


    Protocols:

    1. Make 200 mL stock of M9 (http://www.thelabrat.com/protocols/m9minimal.shtml) (http://www.protocolsonline.com/recipes/media/m9-medium-5x/)
      1. Make M9 salts:
        1. Aliquote 160 ml H2O and add:
          • 12.8 g Na2HPO4-7H2O
          • 3.0 g KH2PO4
          • 0.50 g NaCl
          • 1.0g NH4Cl
        2. Stir until dissolved & Adjust to 200 ml with distilled H2O
        3. Sterilize by autoclaving
        4. Add 4 ml of 20% glucose (or other carbon source) - filter sterilize separately
          • 8 mL of glucose (12.16 g)
          • 32 mL of water
        5. Measure ~140 ml of distilled H2O (sterile)
        6. Add 0.4 ml of 1M MgSO4 (sterile)
        7. Add 20 ul of 1M CaCl2 (sterile)
        8. Adjust to 200ml with distill H2O
        9. Autoclave from b to f first and then mix with M9 salt solution of step i.
        10. Add 40 ml of M9 salts

      Note: M9 media ratios are 16:4:0.4 respectively for MgSO4 solution, M9 salts and glucose. MgSO4+water+agar solution expires 2 weeks after first use.

    2. Melt LB (12 mL) with microwave
    3. Pour M9 and LB into the small plates each with a different thickness:
      • 5 mL
      • 3.5 mL
      • 2.5 mL
      • 1.5 mL
    4. Drop 2.5 uM of 20 uL of FDNA in one corner of each plate
    5. Negative control:
      • blank plate, no M9 or LB
      • Leaving 2 plates without FDNA but with 5 mL of each medium
    6. Observe under UV light & Typhoon Imaging



    Experiment 1.b. Optimal pH of M9 Media for Cell Growth


    Date of Experiment: June 12-13
    Purpose: To determine the effect of pH change on E. coli bacterial growth and autofluorescence on M9 and LB plates.

    Reagents

    • LB Media
    • M9 Media
    • E. coli cells from glycerol stock
    • NaOH (0.2 M)
    • Small agar plates
    • Disposable wire loop


    Protocol:

    1. Create M9 stock media at pH range: control (7), Plate 1 (7.2), Plate 2 (7.6), Plate 3 (8.0), Plate 4 (8.5) by adding volumes of 0.2 M NaOH and checking with a pH meter.
      • Control Plate: No NaOH added, pH measured to be 7.0
      • Plate 1: 300 uL of NaOH added, 7.2 pH
      • Plate 2: 750 uL of NaOH added, 7.6 pH
      • Plate 3: 1500 uL of NaOH added, 8.0 pH
      • Plate 4: 1800 uL of NaOH added, 8.5 pH

      • Note: We poured 2.5 mL of M9 solution out after each plate’s pH was measured, thus the total volume of solution that NaOH is added to gradually decreases by 2.5 mL each time, impacting the concentration of NaOH in each plate

      *Note: pH measured in 40 degrees Celsius solution (M9 solidifies quickly at room temperature). Pour 5 M9 media plates (one for each pH value) at the desired thickness as determined in the previous experiment. (Thickness found to be 2.5 mL of M9 for the small plates) </p>
    2. Using a disposable wire loop, plate E. coli cells in a specific streaking pattern across the plate quadrant for bacteria streaking.
    3. Add FQ substrate spot (2.5 uM, 10uL) onto the FQ quadrant of the plate (bottom left in photo)
      • Positive control: Plate bacteria on unaltered M9 (control plate).
      • Negative control: No bacteria on unaltered plate (see negative quadrant of each plate)
    4. Incubate bacteria at 37 degrees Celsius for 12 hours.
      1. We streaked E coli cells at 11:30 AM, June 13, 2017 and put all plates in the incubator at 11:57 AM on the same day.
      2. We checked on bacterial growth and FQ cleavage using UV and typhoon imaging at 4:00 PM of the same day (1st interval check).
      3. Incubate again overnight.
      4. Check growth and cleavage next morning.
    5. Check bacterial growth and fluorescence of plates using the Typhoon Imager.


        Exp 1.c. Cell Detection Using DNAzyme in M9


        Date of Experiment: June 14-15, 2017
        Purpose: To observe the effectiveness of DNAzyme in M9 with the pH of 8 to detect E. coli colonies. This is within the optimal range mentioned in “A Sensitive DNA Enzyme-Based Fluorescent Assay for Bacterial Detection (Aguirre et al.)

        Materials & Reagents:

        • UV box & Typhoon imager
        • Incubator
        • Small agar plates
        • Disposable wire loop


        • E. Coli cells from glycerol stock (K12 3)
        • DNAzyme (RFD-EC1) 2.245 uM
        • Selection buffer
        • FQ (2.5 uM)
        • NaOH (0.2 M)
        • HCl (0.2 M)
        • M9 solution


        Protocol:

        1. Prepare two M9 plates at pH 8
        2. Streak E. coli cells onto the plate suing a wire loop into 3 quadrants

          3. <img src="http://paste.pics/27B4V" alt="E coli plate wire loop 1">

          • The streaking pattern should look like this.
        3. Prepare DNAzyme SB (5 uL of stock + 5 uL of 2x SB)
        4. Place in incubator (37 degrees celsius) overnight
        5. Drop DNAzyme and FQ into the different quadrants as shown:
          • Quadrant:
            • 1.Cell + DNAzyme (4.454 uL)
            • 2. Cell + FQ (4 uL diluted)
            • 3. Cell only
            • 4. FQ (4 uL) + NaOH (enough to cover FQ ~2 uL)
        6. Afterwards, put in box (don’t expose to light)
        7. For quadrant 4, let FQ be cleaved over time and add HCl (2 uL) to neutralize right before screening.
        8. After ~5 hours, observe plate under UV light and record observations
        9. Analyze fluorescence using the typhoon imager to view any other sources of fluorescence



          10. Experiment 2.a. Differing [NaOH] for Positive Control


          Date of Experiment: June 19-20, 2017
          Purpose:To ensure that NaOH can cleave the FQ substrate so that we have a reliable positive control in future experiments.

          Reagents:

          • NaOH (varying concentrations)
          • FQ substrate (2.5 uM)
          • Eppendorf tubes
          • UV light
          • Typhoon imager


          Protocol:

          1. Add 6uL of FQ substrate to 5 separate Eppendorf tubes.
          2. Dilute the NaOH stock to the appropriate concentrations for use.
          3. Add 4uL of NaOH to each tube with concentrations as described in the table below:
            4. Tube # 1 2 3 4 5
            [NaOH] 0.2 0.4 0.6 0.8 1.0
          4. Let tubes sit for 30 minutes, then observe fluorescence using the UV box in the cold room.
          5. Transfer 10 uL of NaOH+FQ sub as well as 10uL FDNA control onto labeled quadrants of M9 media plates as drawn below:
            6.  <img src="http://paste.pics/27B5T"
 alt="NaOH Plate formations">



          Exp 2.b. Testing DNAzyme Sensitivity


          Date of Experiment: June 22-25 Purpose: Proof of concept that RFD-EC1 probe is specific only to E. coli that express the RNAse I enzyme

          Setup:

          • E. coli K12 + DNAzyme (4.45 uL of 2.245 uM)
          • E. coli K12 (delta RNAseI) + DNAzyme (4.45 uL of 2.245 uM)
          • FDNA (4 uL of 2.5 uM)
          • Nothing
            • Bacteria was streaked on each plate in respective quadrants and grown overnight. next morning: DNAzyme was dropped on cells and FDNA was dropped on 3rd quadrant. The M9 media is pH 8, 2.5 mL.



          Exp 3a Reducing Colony Formation Time


          Date of Experiment: June 29, 2017
          Purpose: To determine the minimum amount of time required for E. coli bacterial growth (minimum colony size) to be detectable through fluorescence on M9 plates using UV light.

          Materials & Reagents:

          • UV box
          • Typhoon imager
          • Incubator
          • 2 M9 plates at pH 7
          • E. Coli cells from glycerol stocks (K12)
          • DNAzyme (RFD-EC1) concentration of 50 pM
          • 2.5 uM FDNA


          Protocol:

          1. Prepare one M9 plate at pH 7
            • Label one plate with quadrants: E. coli + DNAzyme, FDNA, E Coli, DNAzyme
            • Label second plate with DNAzyme + E coli, FDNA, E Coli, DNAzyme
          2. Spread 7.24uL DNAzyme on plates and the second plate quadrant E coli + DNAzyme
          3. Streak E. Coli cells onto both plates
            • Drop 7.24uL DNAzyme onto first plate
            • Drop 20uL FDNA on both plates
              • *make sure FDNA does not come in contact with too much light
          4. Place in incubator (37 degrees celsius)
          5. Do an initial scan after 30min
          6. After ~2 hours, observe plate under UV light and record observations
          7. Analyze possible fluorescence using the typhoon imager (ensure DNAzyme is functioning normally)
          8. Place plate back in incubator and take plate out every 1-2 hours (time dependent on how much fluorescence is visible) to view under UV light. Record time and observations.
          9. Determine the minimum time needed to confirm the presence of E. Coli bacteria.



          Exp 3b DNAzyme Specificity

          Date of Experiment: July 5

          Reagents:

          • DNAzyme (50 pmol)
          • E. coli K12 glycerol stock
          • B. subtilis glycerol stock
          • Brevundimonas diminuta glycerol stock
          • Hafnia alvei glycerol stock
          • Achromobacter xylosoxidans glycerol stock
          • Serratia fonticola glycerol stock
          • Leuconostoc mesenteroides glycerol stock
          • Another bacteria glycerol stock
          • UV lightbox & Typhoon Imager
          • M9 Media plates 4cm diameter

          Protocol:

          1. Label 4 M9 pH 7.0-7.2, 2.5mL plates as depicted.
          2. Streak bacteria in pattern shown with a 10uL pipette tip (exception: for plate 1 use a 2uL pipette tip) @ 4:30pm
          3. Leave plates upside down in a 37 degree Celsius incubator overnight (until 9:30am next morning)
          4. Drop 7.24 uL of 6.9 uM DNAzyme (effective concentration: 50pmol). To ensure less spreading drop 3.62 uL (2x).



          Exp 3c Bacterial Mixture

          Date of Experiment: July 7 Purpose: To determine whether the RFD-EC1 DNAzyme can detect E. coli while in a sample of multiple different species of bacteria.

          Materials/Reagents:

          • E. coli (K12) cells
          • B. subtilis cells
          • Serratia Fonticola cells
          • Brevundimonas diminuta cells
          • New M9 media solution of pH 7.0-7.2
          • Plates (4cm diameter)
          • DNAzyme probe (50 picomol per quadrant)


          Protocol:

          1. Plate 2 plates (one with DNAzyme spread before cell growth, another with DNAzyme dropped after cell growth) with M9 solution and separate into 3 quadrants
            • Negative control of nothing
            • Positive control of E. coli + DNAzyme
            • 4 distinct streaks of E. coli, Serratia Fonticola, B subtilis, and Brevundimonas diminuta
              • Make sure each bacteria streak is in a unique pattern to identify which bacteria strain reacted in the end

            2. <img src="http://paste.pics/27BAU" alt="Specificity Plate Test">

          2. Grow the cells overnight
          3. UV image next morning to ensure cells have grown
          4. For plate with label “DNAzyme drop after”, drop and spread 50 picomoles of DNAzyme probe on the positive control and experiment quadrants
            1. Plate: “DNAzyme to be dropped after"
            2. For that, we'll need to drop a larger volume of DNAzyme so we'll use DNAzyme Eppendorf labelled 2.245 uM that's a larger volume and lighter in color (that's the one we diluted with water)
            3. To get 50 picomoles, dilute 20.46 uL of DNAzyme with 20.46 uL of 2x buffer and pour the total volume of 40.9198 uL over the 2 non-negative control quadrants (3/4 of the plate).
            4. (volume may be a lot but the thing is, I kind of need to flood the surface area of the quadrants since I can't spread with hockey sticks - will shift cells - and I need to cover the entire area since I drew cells in thin lines over a large surface area)
            5. If volume is too large, we can drop half of the mixed solution (20.46 uL) first, let set, and drop the other half again as a second layer
          5. Let sit for 30 minutes to 1 hour for DNAzyme to set
          6. UV and Typhoon image to see which bacteria species cleaved DNAzyme probe
            • See if presence of other bacteria interferes with the fluorescence compared to the positive control
              • If brighter bc other bacterial autofluorescence or dimmer bc of lower concentration of target E. coli bacteria strain



            7. Fluorescence detecting wavelength


            Date of experiment: July 25, 2017 Purpose: To find out which wavelength best detects fluorescence from the cleavage of DNAzyme in the presence of E. coli K12 bacteria

            Reagents/Materials:

            • 13.8 uM DNAzyme
            • 2x selection buffer
            • E. coli K12 and delta RNAse
            • 100uM FDNA
            • 2 M9 plates
            • 5 Eppendorf tubes
            • UV box
            • Typhoon imager


            Protocol:

            1. Grow K12 and delta RNAse in liquid culture until both reach a count of 10^8 cells/mL
            2. Label 2 plates with quadrants: K12, delta RNAse, negative, FDNA
            3. In one eppendorf tube, insert 11.45 uL 2xSB, 10uL K12 cells, 1.45uL DNAzyme
            4. Repeat step 3 in a separate eppendorf tube but instead inserting 10uL delta RNAse *** make sure to add DNAzyme last (SB may cause unintentional cleavage)
            5. Place both eppendorf tubes in an incubator for at least 30 minutes
            6. Remove tubes from incubator and drop 10uL from both tubes onto its labeled quadrant on the plate letting it slightly spread
            7. Perform a 1:10 dilution of FDNA in an eppendorf tube using sterile water and drop 2uL onto its quadrant
            8. Image under UV and typhoon to observe fluorescence



            Optimal DNAzyme Concentration


            Date of experiment: July 25, 2017 Experiment done by: Audrey
            Purpose: After discovering the optimal number of bacterial cells needed for visible DNAzyme cleavage, this experiment will find the minimal number/concentration of DNAzyme to show distinguishable fluorescence

            Reagents/materials:

            • 13.8 uM DNAzyme
            • 2x selection buffer
            • E. coli K12
            • 100uM FDNA
            • 2 M9 plates
            • 5 Eppendorf tubes
            • UV box
            • Typhoon imager

                • Protocol:

                1. Grow K12 in liquid culture until it reaches a cell count of 10^8 cells/mL
                2. Label eppendorf tubes: 10pmol DNAzyme, 20pmol DNAzyme, 30pmol DNAzyme, 40pmol DNAzyme, FDNA In 4 separate eppendorf tubes, drop 10uL of cells and the following amount of reagent:
                  • 10pmol DNAzyme: 10.73uL SB + 0.75uL DNAzyme
                  • 20pmol DNAzyme: 11.45uL SB + 1.45uL DNAzyme
                  • 30pmol DNAzyme: 12.17uL SB + 2.174uL DNAzyme
                  • 40pmol DNAzyme: 12.90uL SB + 2.90uL DNAzyme

                  3. *** make sure to add DNAzyme last (SB may cause unintentional cleavage)

                3. Place eppendorf tubes in an incubator for at least 30 minutes
                4. Label 2 plates with quadrants:
                  • 10pmol, 20pmol, DNAzyme, FDNA
                  • 30pmol, 40pmol, DNAzyme, FDNA
                5. Remove tubes from incubator and drop 10uL from tubes onto its labeled quadrant on the plate letting it slightly spread
                6. Drop 5uL DNAzyme separately onto its quadrant (to check for autofluorescence)
                7. Perform a 1:10 dilution of FDNA in an eppendorf tube using sterile water and drop 2uL from the tube onto the plate with the lower cell count. Drop 4uL onto plate with the higher cell count.
                8. Image under UV and typhoon to observe fluorescence



                Quantifying Autofluorescence


                Date of experiment: July 27, 2017 Purpose: It is unsure whether strong fluorescence is partially due to autofluorescence generated from cells and/or the DNAzyme. This experiment will gather numerical data on fluorescence to determine how much autofluorescence exists in different concentrations of bacteria.


                Reagents/materials:

                • 13.8 uM DNAzyme
                • 2x selection buffer
                • E. coli K12 (10^8 cells/mL) liquid culture
                • 2 M9 plates
                • 5 Eppendorf tubes
                • UV box & Typhoon imager


                Protocol:

                1. Grow K12 in liquid culture until it reaches a cell count of 10^8 cells/mL
                2. Dilute liquid culture to 10^6 cells/mL and 10^4 cells/mL
                3. Take 10uL of 10^8 cells/mL and place into a new eppendorf tube. Do the same with 10^6, 10^4 cells/mL
                4. Pipette 11.45uL of selection buffer into each tube and 1.45uL DNAzyme. *** make sure to add DNAzyme last (SB may cause unintentional cleavage)
                5. Place eppendorf tubes in an incubator for at least 30 minutes
                6. Label 3 plates with quadrants: DNAzyme + cells, cells, DNAzyme, negative
                7. Label each plate so it corresponds to one of the three different cell counts (10^6, 10^4, 10^2 cells)
                8. Drop 10uL of the liquid cultures with different concentrations into the “cells” quadrant in the plate that’s labeled with its cell count
                9. Drop 1.45uL DNAzyme separately onto its quadrant and let it spread
                10. Remove tubes from incubator and drop 10uL from tubes onto its labeled quadrant on the plate letting it slightly spread
                11. Image under UV and typhoon to observe fluorescence



                Fluorescing Colonies


                Date of experiment: July 28, 2017 Purpose: Before performing the timepoint experiment, it must be confirmed whether the DNAzyme will fluoresce when dropped onto a colony of E.coli K12


                Reagents/materials:

                • 13.8 uM DNAzyme
                • 2x selection buffer
                • E. coli K12 (10^8 cells/mL) liquid culture
                • Delta RNAse (10^8 cells/mL) liquid culture
                • 2 M9 plates
                • 1 Eppendorf tube
                • UV box & Typhoon imager


                Protocol:

                1. Streak both bacteria onto separate plates labeled: E.coli K12 and delta RNAse
                2. Place in incubator and let it grow for a day (~20 hours) until isolated colonies form and are noticeable
                3. In an eppendorf tube, add 1.45uL 2x selection buffer and 1.45uL DNAzyme
                4. Take plates out of incubator and take 1.45uL from the eppendorf to drop on top of individual colonies in the E.coli K12 plate
                5. Repeat step 4 for the delta RNAse plate
                6. Place both plates in the incubator and set it rest for ~20min
                7. Remove plates from incubator and image under UV and typhoon for observations



                DNAzyme Autofluorescence

                Date of experiment: July 31, 2017 Purpose: As the DNAzyme shows excessive autofluorescence, it was important to see how much DNAzyme could be added to produce the least amount of autofluorescence, but the most cleavage in the presence of E. coli K12.


                Reagents/materials:

                • 13.8 uM DNAzyme
                • 2x selection buffer
                • E. coli K12 (10^8 cells/mL) liquid culture
                • 2 M9 plates
                • 6 Eppendorf tubes
                • UV box
                • Typhoon imager

                    • Protocol:

                    1. Dilute DNAzyme using liquid M9 media in 4 separate eppendorf tubes to obtain the following amounts: 5, 10, 20, 30, 40 pmol
                    2. Drop 20 uL onto quadrants and spread evenly using a hockey stick.
                    3. Let the DNAzyme set for a few minutes then image to observe autofluorescence
                    4. Streak bacteria onto each quadrant to grow colonies
                    5. Place in incubator overnight
                    6. Drop FDNA (positive control) and FQ (negative control) onto its quadrant and image to observe fluorescence

                    New DNAzyme Stock

                    Bacterial Autofluorescence


                    Purpose: To explore the effect bacterial autofluorescence may be having on experiments, to quantify bacterial autofluorescence so as to minimize it in future experiments.

                    Setup:

                    • Eppendorf tubes: (4)
                    • E. coli K12
                    • Bacterial Spreading Sticks (4)
                    • M9 Plates (4)
                    • UV Imager
                    • Typhoon Imager


                    Protocol:

                    1. Make 4 dilutions for E. coli K12. This will be 10^9 cells/mL, 10^7 cells/mL, 10^6 cells/mL, and 10^5 cells/mL (4 tubes in total).
                    2. Transfer 30uL of each of these to a new separate Eppendorf tube.
                    3. Image under UV immediately and after 30 minutes of incubation.
                    4. Before the 1hr mark, plate and spread (with hockey stick) all of these bacteria separately.
                    5. Incubate plates overnight at 37 degrees Celsius upside down.
                    6. The next morning (SHOULD BE EXACTLY 16 HOURS AFTER, PLAN ACCORDINGLY), image under UV and Typhoon.



                    DNAzyme Autofluorescence


                    Date: August 21, 2017 Purpose: To quantify and minimize the amount of autofluorescence produced from the DNAzyme.

                    Setup:

                    • Plates from the previous experiment can be used (the empty quadrants (8))
                    • DNAzyme
                    • 2X SB
                    • UV Imager
                    • Typhoon Imager


                    Protocol:

                    1. After completing the bacterial experiment (2), utilize the empty quadrants (4).
                    2. Make DNAzyme dilutions: 40pmol/5uL,30pmol/5uL, 20pmol/5uL, 10pmol/5uL. = 8pmol/uL, 6pmol/uL, 4pmol/uL, 2pmol/uL
                    3. Put 5uL of these into separate Eppendorf tubes. Image under UV immediately.
                    4. Plate the 5uL in the empty quadrants of the bacterial plates. Incubate right side up for 1hr at 37 degrees Celsius and image under UV and with the Typhoon.Incubate for another hour and image again under Typhoon.



                    Optimal Response


                    Date: August 21, 2017 Purpose: To generate the optimal response for the DNAzyme to detect E. coli K12 while minimizing all autofluorescence.

                    Setup:

                    • DNAzyme: pmol/5uL, pmol/5uL, pmol/5uL, pmol/5uL
                    • E. coli K12 cells on plates from experiment (2)
                    • E. coli K12 delta RNase cells from experiment (2)
                    • UV Imager & Typhoon Imager


                    Protocol:

                    1. On the same day as the bacterial autofluorescence experiment Day 2, once results are obtained for that experiment, do this experiment.
                    2. Drop the 5uL of each DNAzyme amount on individual colonies of E. coli K12 and E. coli K12 delta RNase for each bacterial number. BE SURE TO KEEP TRACK HOW MUCH DNAZYME IS PUT ON WHICH COLONY!
                    3. Incubate the plates right side up for 1 hr, then image under UV and Typhoon Image.
                    4. Image again at 2 hrs with the Typhoon Imager.


                    DNAzyme Specificity

                    Date: August 21, 2017 Purpose: To verify the specificity of the DNAzyme solely for E. coli K12, and to show the inability to cleave against other bacterial species.

                    Setup:

                    • E. coli K12 (10^8 cells/mL)
                    • E. coli K12 delta RNase (10^8 cells/mL)
                    • Bacillus subtilis (10^8 cells/mL)
                    • Hafnia alvei (10^7 cells/mL)
                    • Achromobacter xylosoxidans (10^7 cells/mL)
                    • Serratia fonticola (10^7 cells/mL)
                    • DNAzyme (A)
                    • 2X SB
                    • FDNA (10uM)
                    • M9 plates
                    • UV Imager & Typhoon Imager


                    Protocol:

                    1. Grow overnight liquid cultures in M9 media of each bacterial species.
                    2. Plate 30uL of cells (10^4 cells) on each quadrant and grow for 16 hours at 37 degrees Celsius. Remember to put cell cultures back in the fridge after use!
                    3. After the 16 hours, drop the 5uL of DNAzyme on an individual colony of each bacterial species.
                    4. Incubate right side up for 1hr and UV image and Typhoon image.
                    5. Incubate for another hour and Typhoon Image.


                    Tracking Fluorescence Over Time

                    Date: August 21, 2017 Purpose: To quantify DNAzyme fluorescence over time and determine whether the addition of selection buffer impacts fluorescence results.

                    Setup:

                    • E. coli K12
                    • DNAzyme (40pmol/5uL)
                    • 2X SB
                    • Typhoon Imager
                    • UV Imager
                    • M9 plates

                        • Protocol:

                        1. Incubate E. coli cells at 37 degrees Celsius until a density of /mL
                        2. Plate 30uL of cells and let grow for 16 hours at 37 degrees Celsius.
                        3. Drop 5uL of DNAzyme (pmol) + 5uL 2xSB on a colony.
                        4. Drop 5uL of DNAzyme (pmol) + 5uL water on another separate colony.
                        5. UV image immediately. Typhoon Image Immediately.
                        6. Typhoon Image after 30 minutes, 1hr, 2hrs, 3hrs, 4hrs, and 5 hrs.
                        7. Label all results and post them here.



                        Experiments Redone Without Selection Buffer


                        Date of Experiment: September 28 Purpose: To prove the specificity of the DNAzyme and supply appropriate measurements for data analysis.

                        Materials:

                        • M9 plates
                        • E. coli K12 & E. coli K12 delta RNase
                        • B. subtilis
                        • A. xylosoxidans
                        • H. alvei
                        • S. fonticola


                        Protocols:

                        1. Culture all bacteria in liquid M9 media overnight
                        2. Look at the OD for bacteria and calculate cell density (around 10^8-10^9 cells/mL).
                        3. Dilute cells as necessary and plate 30uL onto a quadrant of a plate. 3 quadrants bacteria, one quadrant empty (for negative control). *made replicates to verify results
                        4. Put the plates in the incubator upside down for 16 hrs exactly
                        5. An hour before removing the plates from the incubator, prepare the DNAzyme (the optimal amount per 5uL).
                        6. Drop the 5uL of DNAzyme onto each bacterial quadrant as and one control quadrant
                        7. Incubate with the DNAzyme for 1 hour and then image with the typhoon imager.


                        Enzyme Kinetics & 96 Well Plate


                        Without Selection Buffer

                        Date: September 25

                        Materials:

                        • 96-well plate (black-clear bottom)
                        • E. coli K12 (at least at 10^7cells/mL or 10^4cells/uL)
                        • Liquid M9
                        • Autoclaved distilled water
                        • DNAzyme (RFD-EC1)


                        Protocol:

                        1. Grow a liquid culture of E. coli bacteria in liquid M9 overnight in a shaker incubator
                        2. Pipette 90uL of liquid M9 from A2-7 and C2-7
                        3. Dilute with liquid M9 (if necessary) to obtain a cell concentration of 10^7cells/mL or 10^4cells/uL
                        4. Transfer 100uL of cells to wells A1 and C1
                        5. Do a serial dilution and transfer 10uL across the wells (e.g. from A1 to A2, A2 to A3, etc)
                          • once you’ve reached row 6, take out 10uL but DO NOT put into row 7
                        6. At this point, 90uL should be the volume in every well used for this experiment
                        7. Dilute using sterile distilled water (if necessary) to reach a concentration of 4uM
                        8. Using a multichannel pipette, drop 10uL of DNAzyme into the upper row (A1-7)
                        9. Scan immediately in plate reader to observe kinetics


                        With Selection Buffer (Replicates)

                        Date: October 5

                        Materials:

                        • Everything else listed in previous experiment (including selection buffer)


                        Protocol:

                        1. Repeat all steps in previous experiment on a new plate doing triplicates to verify data and create error bars (used rows A to F)





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