• 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.