WETLAB EXPERIMENTS

The PCR system was formulated according to the table below

Reagent	Volume(μL)
DNA	1
Primer-F	0.5
Primer-R	0.5
2X Master Mix	5
ddH2O	3

Put in PCR instrument, PCR program

Temperature	Time
98°C	30s
98°C	10s
65°C	30s
72°C	21s
72°C	10min

1) Weigh 0.5g agarose and add to 50ml 1X TAE.

2) Microwave until completely melted.

3) Cool to 50 ° C to 60 ° C and add 3μL DNA stain.

4) Inverted board.

5) After cooling, sample in sequence.

6) 130V 30min.

7) After the process is completed, place the gel under a UV lamp, excise the portion of the gel containing the target fragment, and place the excised gel into a 1.5 mL EP tube.

1) Add 200 μL of PN to the colloid and incubate in a water bath at 50°C for 10 minutes.

2) Add 500 μL of BL to the chromatography column and place it in a centrifuge at 12,000 rpm for 1 minute. Discard the eluate.

3) Transfer the liquid in the EP tube to the chromatography column and let it stand at room temperature for 2 minutes. Then place it in the centrifuge at 12,000 rpm for 1 minute and discard the eluate.

4) Add 600 μL of PW to the column and place it in the centrifuge at 12,000 rpm for 1 minute. Discard the eluate.

5) Repeat the previous step once.

6) Place the tube in the centrifuge at 12,000 rpm for 2 minutes without any liquid, then open the lid and let it stand for 5 minutes.

7) Place the chromatography column in a new EP tube and add 30 μL of ddH2O to the column. Let it stand for 2 minutes and then place it in the centrifuge at 12,000 rpm for 2 minutes. Collect the eluate.

8) Add the eluate back to the chromatography column and place the system in the centrifuge at 12,000 rpm for 2 minutes. Discard the chromatography column and keep the EP tube.

1) Prepare the enzyme digestion system

2） 37℃ 40min

1）Prepare the connection system（10μL）

2） 4℃ for the whole night

1) Add 10 μL of plasmid to the EP tube.

2) Add 20 μL of competent cells to the EP tube.

3) Place the system on ice and let it stand for 30 minutes.

4) After completion, quickly transfer the system to a water bath and perform a heat shock at 42°C for 35 seconds.

5) Add 500 μL of LB culture medium to the system and place the entire system in a shaker for 1 hour.

6) After the previous section is completed, place the entire reaction system on the centrifuge and centrifuge it at 4000 rpm for 5 minutes.

7) After completion, discard the supernatant of the system, 450 μL, and then use the remaining liquid to resuspend and mix the precipitate before coating the plate.

1) Add 500 μL of medium and 0.5 μL of ampicillin to a 1.5 mL centrifuge tube, pick a single colony, and incubate at 37°C with shaking at 220 rpm for 2-3 hours.

2) Inoculate the bacteria from the 1.5 mL centrifuge tube into a 50 mL centrifuge tube containing 20 mL of medium, add 20 μL of ampicillin, and incubate at 37°C with shaking at 220 rpm. Measure the OD value using a microplate reader. When the OD value reaches 0.5-0.8, take out the bacterial culture and allow it to cool. Add 20 μL of IPTG (concentration 0.42 M), and induce at 18°C with shaking at 220 rpm for 24 hours at low temperature.

3) After 24 hours, centrifuge at 4000 rpm for 5 minutes, resuspend in 1 mL of HEPES Buffer to obtain a protein suspension, and store at -80°C.

1) Lysate Preparation: The protein suspension is sonicated on ice at 20–30% power for 5 minutes, then centrifuged at 12,000 rpm for 10 minutes. The target protein remains in the supernatant.

2) Magnetic Bead Activation: Transfer His-Tag magnetic beads to a 1.5 mL tube, add 600 µL of HEPES Buffer, and mix thoroughly using a 1 mL pipette. Separate the beads from the buffer using a magnetic stand and discard the buffer. Repeat this washing process three times. After the final wash, remove all buffer.

3) Protein Binding: Add the protein supernatant to the washed magnetic beads and incubate at 4 °C for 3 hours with gentle mixing to facilitate binding.

4) Elution: Elute the bound protein using elution buffers containing imidazole at concentrations of 50 mM, 100 mM, 300 mM, and 500 mM.

5)Sample Collection: Retain the eluted fractions from the 100 mM and 300 mM imidazole elutions for further analysi

1) Prepare a 1 μmol/L protein solution in HEPES buffer (or appropriate buffer).

2) Add 100 μL of the protein solution to each well of a 96-well black microplate.

3) Place the plate in a pre-equilibrated microplate reader and measure fluorescence intensities at 420 nm and 485 nm under different temperatures (20 °C, 25 °C, 30 °C, 35 °C, 40 °C).

4) Add Furoxan to each well to a final concentration of 100 μM.

5) Immediately monitor fluorescence changes at 420 nm and 485 nm under each temperature condition.

6) Calculate the fluorescence ratio (F420/F485) over time.

1) Prepare 1 μmol/L protein solutions in buffers with different pH values (7.0, 7.2, 7.4, 7.6, 7.8, 8.0). Use appropriate buffers (e.g., HEPES, Tris, phosphate) adjusted to target pH.

2) Add 100 μL of each pH-adjusted protein solution to a 96-well black microplate.

3) Measure initial fluorescence at 420 nm and 485 nm.

4) Add Furoxan to a final concentration of 100 μM in each well.

5) Record fluorescence changes at both wavelengths and calculate the F420/F485 ratio.

1) Add 100 μL of 1 μmol/L protein solution to each well of a 96-well black microplate.

2) Measure baseline fluorescence at 420 nm and 485 nm.

3) Add Furoxan to final concentrations of 0, 1, 10, and 100 μM (using serial dilutions if needed).

4) Incubate for 5–10 minutes and record fluorescence intensities.

5) Calculate the fluorescence ratio (F420/F485) for each Furoxan concentration.

1) Add 100 μL of 1 μmol/L protein solution to each well of a 96-well black microplate.

2) Record initial fluorescence at 420 nm and 485 nm.

3) Add different analytes (Furoxan, CO, H₂O₂, NaNO₂, NaHS) to a final concentration of 100 μM in separate wells.

4) Incubate for 10–15 minutes at room temperature.

5) Measure fluorescence intensities at both wavelengths and calculate F420/F485 ratios.

6) Include a negative control (buffer only) and positive control (Furoxan) in each experiment.

Quickly remove the cryovial from liquid nitrogen and thaw it in a 37°C water bath. Transfer the cell suspension to a 15 mL centrifuge tube containing 5 mL of pre-warmed complete medium. Centrifuge at 1000 rpm for 5 minutes. Discard the supernatant, resuspend the cell pellet in fresh medium, and seed it into a T25 culture flask. Gently mix to ensure even distribution.

a) Passage the cells when they reach 80-90% confluency. Avoid overgrowth.

b) Aspirate and discard the old medium.

c) Gently rinse the cells with 3-5 mL of DPBS (Dulbecco's Phosphate-Buffered Saline) to remove residual serum and dead cells.

d) Aspirate the DPBS and add 1-2 mL of 0.25% Trypsin-EDTA (for a T25 flask). Gently swirl to ensure even coverage of the cell layer.

e) Incubate at 37°C for 1-2 minutes, or observe under a microscope. Proceed immediately when the cells round up and gaps appear between them.

f) Add 2-3 volumes of complete medium (containing FBS to inhibit trypsin activity). Gently pipette the solution to dislodge all cells and form a single-cell suspension.

g) Transfer the cell suspension to a centrifuge tube and centrifuge at 1000 rpm for 5 minutes.

h) Discard the supernatant and resuspend the cell pellet in fresh medium.

i) Passage the cells at a 1:5 to 1:10 split ratio (e.g., from a T25 flask to a T75 flask, or from one T75 flask to three T75 flasks).

j) Add fresh medium, gently swirl to mix, and return the flask to the incubator.

1) Take a sterile 1.5 mL centrifuge tube and sequentially add the following:

• Ultrapure water: to a final volume of 125 μL

• Plasmid DNA mixture: 3.0 μg (diluted with ultrapure water)

• 2.5M CaCl₂: 12.5 μL

Gently mix.

2) Take another sterile 1.5 mL centrifuge tube and add 125 μL of 2× HBS solution.
Using a pipette, slowly add the DNA/CaCl₂ mixture dropwise into the 2× HBS solution while gently blowing to mix (add slowly, drop by drop).

3) After adding, immediately pipette up and down gently 3-5 times to mix. The solution should appear slightly turbid without visible large aggregates.

4) Incubate at room temperature for 15-20 minutes. During this time, fine DNA-calcium phosphate complexes will form, and the solution will become slightly clearer.

5) Evenly add the 250 μL transfection complex mixture dropwise into the cell culture medium in the 6-well plate, gently rocking the plate while adding.

6) Return the cell culture plate to the 37°C, 5% CO₂ incubator.

7) After 6-8 hours (or following overnight incubation), observe under a microscope. The cells should be covered with fine black particles. At this point, replace the medium with fresh complete medium (2 mL/well). This step reduces toxicity and improves cell viability.

8) Continue culturing for 48-72 hours, then collect the supernatant containing viral particles (if performing virus packaging).

1) Solution Preparation:

• Using artificial plasma, dilute the Furoxan stock solution to 2× the target final concentration. Specifically:

- 0 μM group: Artificial plasma + corresponding volume of solvent (e.g., DMSO).

- 200 μM group: Dilute to 400 μM (2× the final concentration of 100 μM).

- 1000 μM group: Dilute to 2000 μM (2× the final concentration of 500 μM).

- 2000 μM group: Dilute to 4000 μM (2× the final concentration of 1000 μM).

• Using artificial plasma, dilute the NO fluorescent probe protein to its working concentration (as recommended in the probe manual).

2) Sample Loading and Reaction:

• Take four 1.5 mL centrifuge tubes and label them as 0, 100, 500, and 1000 μM.

• To each tube, add 50 μL of the corresponding 2× Furoxan solution.

• Then, add 50 μL of the diluted probe protein working solution to each tube.

• Vortex immediately to mix thoroughly and start the timer.

3) Incubation and Detection

• Transfer the entire reaction mixture to a black 96-well plate (or cuvette).

• Incubate the reaction system at 37°C in the dark for a specific duration (e.g., 30 minutes, or as required by the experimental design).

• Place the plate into the blue light excitation instrument.

• Set the excitation/emission wavelengths.

• Detect and record the fluorescence intensity of each well.