1. In a PCR tube, add 25 µL 2X Phusion Plus PCR Master Mix.
2. Add template DNA.
3. Nanodrop DNA result to find ng/µL value
4. Kit can take between 5-100 ng of genomic DNA.
   1. x µL of Template DNA yields about 100 ng of genomic DNA.
   2. x µL of Template DNA yields about 100 ng of genomic DNA.
5. Add 10 µL GC enhancer.
6. Add 0.5 µL PCR forward primer. 50 µM stock x 0.5 µL = 0.5 µM working x 50 µL reaction volume.
7. Add 0.5 uL PCR backward primer. 50 µM stock x 0.5 µL = 0.5 µM working x 50 µL reaction volume.
8. Add 4 µL of water to bring the reaction up to 50 µL.
9. Perform PCR: Thermocycler settings
   1. Initial Denaturation: 98°C for 30 sec.
   2. Denaturation: 98°C for 10 sec.
   3. Annealing: 65°C for 10 sec.
   4. Extension: 72°C for 30 sec.
   5. Cycle between denaturation, annealing, and extension for 30 cycles.
   6. Final extension: 72°C for 5 min.
   7. Infinite hold.

1. Add a 1:1 volume of Binding Buffer to the PCR product.
2. Add 50 µL of Binding Buffer for the 50 µL PCR reaction.
3. If the color of the solution is orange or violet, add 10 μL of 3 M sodium acetate, pH 5.2 solution and mix. The color of the mix will become yellow.
4. Add a 1:2 volume of 100% isopropanol aka add 50 μL of isopropanol and mix thoroughly.
5. Transfer up to 800 μL of the solution to the GeneJET purification column.
6. Centrifuge for 30-60 s. Discard the flow-through.
7. Add 700 μL of Wash Buffer to the GeneJET purification column.
8. Centrifuge for 30-60 s. Discard the flow-through and place the purification column back into the collection tube.
9. Centrifuge the empty GeneJET purification column for an additional 1 min to completely remove any residual wash buffer.
10. Transfer the GeneJET purification column to a clean 1.5 mL microcentrifuge tube.
11. Add 30 μL of Nuclease Free Water to the center of the GeneJET purification column membrane and centrifuge for 1 min.
12. Discard the GeneJET purification column and store the purified DNA at -20 °C.
13. Use NanoDrop spectrophotometer to quantify amount of extracted DNA

1. Mix 0.5 g of agarose with 50 mL of 1x TAE buffer in a 100-250 mL Erlenmeyer flask
2. Heat up the solution until it turns clear
3. Set microwave and check every 30 seconds
4. Be careful because it boils and overflows easily
5. Let solution cool down until it reaches 65°C
6. Check with thermometer gun or wait until you can hold the flask for approximately 10 seconds
7. Once the solution measures 65°C, add 6 µL of SybrSafe in and gently shake the flask to mix the solution
8. Put in the gel comb and pour the solution into a gel chamber
9. Wait for the gel to solidify. Once solidified, fix the gel’s position so that the wells of the gel are at the end of the chamber and DNA runs to red
10. Pour used 1x TAE buffer into the gel chamber evenly to completely cover the gel
11. In a PCR tube, mix 5 µL of DNA and 1 µL of purple loading dye using a micropipette by pipetting up and down
12. Add the mixed 6ul solution into each well
    1. Label the wells in your notebook before actually adding solutions to wells
    2. Be careful and make sure the micropipette tip does not puncture the agarose gel
13. Add 10 µL of 2-log ladder into an empty well
14. Connect the electrodes by closing the gel chamber and connecting the to the power supply
15. Set power supply for 90 volts and 45 minutes
16. Turn on the power supply and make sure bubbles are rising on the sides of the gel chamber
17. Once gel electrophoresis is completed, put the gel under UV (ultraviolet) light to compare the bands of DNA to the bands of the ladder
    1. By figuring out the base pair lengths of DNA solutions, you can confirm the presence of correct inserts and vectors in the solution

1. Mix 0.75 g of agarose with 50 mL of 1x TAE buffer in a 100-250 mL Erlenmeyer flask
2. Heat up the solution until it turns clear
3. Set microwave and check every 30 seconds
4. Be careful because it boils and overflows easily
5. Let solution cool down until it reaches 65°C
6. Check with thermometer gun or wait until you can hold the flask for approximately 10 seconds
7. Once the solution measures 65°C, add 6 µL of SybrSafe in and gently shake the flask to mix the solution
8. Put in the gel comb and pour the solution into a gel chamber
9. Wait for the gel to solidify. Once solidified, fix the gel’s position so that the wells of the gel are at the end of the chamber and DNA runs to red
10. Pour used 1x TBE buffer into the gel chamber evenly to completely cover the gel
11. In a PCR tube, mix 5 µL of DNA and 1 µL of purple loading dye using a micropipette by pipetting up and down
12. Add the mixed 6ul solution into each well
    1. Label the wells in your notebook before actually adding solutions to wells
    2. Be careful and make sure the micropipette tip does not puncture the agarose gel
13. Add 10 µL of 2-log ladder into an empty well
14. Connect the electrodes by closing the gel chamber and connecting the to the power supply
15. Set power supply for 90 volts and 45 minutes
16. Turn on the power supply and make sure bubbles are rising on the sides of the gel chamber
17. Once gel electrophoresis is completed, put the gel under UV (ultraviolet) light to compare the bands of DNA to the bands of the ladder
    1. By figuring out the base pair lengths of DNA solutions, you can confirm the presence of correct inserts and vectors in the solution

1. Protein-Aptamer Binding Reaction

• Incubate at 37 Celsius for 15 minutes

2. Ligation Step

• Incubate at 37 Celsius for 5 minutes

• Incubate at 37 Celsius for 5 minutes

V - Cas12a Reaction Setup

• Chemically competent NEB 5-alpha Competent E. coli cells (Stored at -80°C)

1. Thaw a tube of NEB 5-alpha Competent E. coli cells on ice until the last ice crystals disappear.
2. Mix gently and carefully pipette 50 ul of cells into a transformation tube on ice.
3. Add 1-5 ul containing 100 pg-1 ugZ of plasmid DNA to the cell mixture. Carefully flick the tube 4-5 times to mix cells and DNA. Do not vortex.
4. Place the mixture on ice for 30 minutes. Do not mix.
5. Heat shock at exactly 42°C for exactly 30 seconds.Do not mix.
6. Place on ice for 5 minutes. Do not mix.
7. Pipette 950 ul of room temperature SOC into the mixture.
8. Place at 37°C for 60 minutes. Shake vigorously (250 rm) or rotate.
9. Warm selection plates to 37°C.
10. Mix the cells thoroughly by flicking the tube and inverting, then perform several 10-fold serial dilutions in SOC.
11. Spread 50-100 ul of each dilution onto a selection plate and incubate overnight at 37°C. Alternatively, incubate at 30°C for 24-36 hours or 25°C for 48 hours.

1. Pipette 5mL of Luria Broth into a culture tube
2. Pipette 5uL of 1000x antibiotic (spectinomycin) into the culture tube
3. Use a sterile inoculation loop to pick up a colony and stir into the culture tube.
4. Incubate the culture at 37 Celsius for 24 hours while shaking

1. Resuspend pellet cells in 250ul Resuspension solution. Transfer the cell suspension to microcentrifuge tube.
2. Add 250 uL of Lysis solution, invert until solution is clear.
3. Add 350ul of Neutralization solution, invert tube 4-6 times.
4. Centrifuge microcentrifuge tube for 5 min at 15600 rpm.
5. Pipet supernatant to the GeneJET spin column, avoid disturbing the white precipitate
6. Centrifuge the GeneJET column for 1 min at 15600 rpm
7. Add 50 uL Wash solution and centrifuge for 1 min at 15600 rpm
8. Discard flow through and place column back in collection tube
9. Add 50 uL Wash solution and centrifuge for 1 min at 15600 rpm
10. Discard flow through and placed column back in collection tube
11. Centrifuge 1 min to remove residual Wash solution.
12. Transfer GeneJET spin column to 1.5 mL centrifuge tube and discard collection tube
13. Add 30 uL of Elution buffer to the center of GeneJET spin column membrane to elute plasmid DNA
14. Incubate 2 minutes at room temperature
15. Centrifuge column in centrifuge tube for 2 minutes
16. Discard column and store purified plasmid DNA at -20°C

• Room temperature Protect from light Kit Contents for Viability Kit, L13152
• SYTO 9 dye (Component A), stabilized as a solid in 10 sealed applicator pipets
• Propidium iodide (Component B), as a solid in 10 sealed applicator pipets
• BacLight mounting oil (Component C), 10 mL, for bacteria immobilized on membranes. The refractive index at 25°C is 1.517 ± 0.003. DO NOT USE FOR IMMERSION OIL.
• For use of the applicator pipets provided in kit L13152, snip off the sealed ends and dissolve the contents in deionized water, as described in the protocols below.

Culture Conditions and Preparation of Bacterial Suspensions Note: Care must be taken to remove traces of growth medium before staining bacteria with these kit reagents. The nucleic acids and other media components can bind the SYTO 9 and propidium iodide dyes in unpredictable ways, resulting in unacceptable variations in staining. A single wash step is usually sufficient to remove significant traces of interfering media components from the bacterial suspension. Phosphate wash buffers are not recommended because they appear to decrease staining efficiency.

General Protocol:

1. Grow 30 mL cultures of either Escherichia coli or Staphylococcus aureus to late log phase in nutrient broth (e.g., DIFCO catalog number 0003-01-6).
2. Concentrate 25 mL of the bacterial culture by centrifugation at 10,000 × g for 10–15 minutes.
3. Remove the supernatant and resuspend the pellet in 2 mL of 0.85% NaCl or appropriate buffer.
4. Add 1 mL of this suspension to each of two 30–40 mL centrifuge tubes containing either 20 mL of 0.85% NaCl or appropriate buffer (for live bacteria) or 20 mL of 70% isopropyl alcohol (for killed bacteria).
5. Incubate both samples at room temperature for 1 hour, mixing every 15 minutes. 1.6 Pellet both samples by centrifugation at 10,000 × g for 10–15 minutes.
6. Resuspend the pellets in 20 mL of 0.85% NaCl or appropriate buffer and centrifuge again as in step 1.6.
7. Resuspend both pellets in separate tubes with 10 mL of 0.85% NaCl or appropriate buffer each.
8. Determine the optical density at 670 nm (OD670) of a 3 mL aliquot of the bacterial suspensions in glass or acrylic absorption cuvettes (1 cm pathlength).
9. For suggested concentrations of E. coli or S. aureus suspensions, please refer to the section appropriate for your instrumentation: fluorescence microscope, fluorometer, fluorescence microplate reader or flow cytometer.

Staining Bacterial Suspensions with Kit L13152

1. Adjust the E. coli suspensions (live and killed) to 4 × 108 bacteria/mL (about 0.12 OD670) or the S. aureus suspensions (live and killed) to 4 × 107 bacteria/mL (~0.60 OD670). S. aureus suspensions typically should be 10-fold less concentrated than E. coli when using a fluorescence microplate reader.
2. Mix five different proportions of E. coli or S. aureus (Table 3) in 16 × 125 mm borosilicate glass culture tubes.
3. Prepare a 2X working solution of the LIVE/DEAD BacLight staining reagent mixture by dissolving the contents of one Component A pipet (containing yellow-orange solids) and one Component B pipet (containing red solids) in a common 5 mL–volume of filter-sterilized dH2O.
4. Pipet 100 µL of each of the bacterial cell suspension mixtures into separate wells of a 96-well flat-bottom microplate. We recommend that you prepare samples in triplicate. The outside wells (rows A and H and columns 1 and 12) are usually kept empty to avoid spurious readings.
5. Using a new tip for each well, pipet 100 µL of the 2X working stain solution (from step 8.3) to each well and mix thoroughly by pipetting up and down several times. 8.6 Incubate the sample at room temperature in the dark for 15 minutes.

Fluorescence Measurement and Data Analysis

1. With the excitation wavelength centered at about 485 nm, measure the fluorescence intensity at a wavelength centered at about 530 nm (emission 1; green) for each well of the entire plate.
2. With the excitation wavelength still centered at about 485 nm, measure the fluorescence intensity at a wavelength centered about 630 nm (emission 2; red) for each well of the entire plate.
3. Analyze the data by dividing the fluorescence intensity of the stained bacterial suspensions (Fcell) at emission 1 by the fluorescence intensity at emission 2. Ratio G/R = F F cell,em1 cell,em2 9.4 Plot the RatioG/R versus percentage of live cells in the E. coli suspension (Figure 2).