1 L formula:

Procedure:

1. Prepare the liquid and solid medium according to the system above
2. Only solid medium needs to be supplemented with AGAR powder, 15–20 g/L.
3. 121 ℃, 15–20 minutes.
4. Add 100 μLof 50 mg/mL Kana solution to every 100 mL medium for solid media.

To prepare 10 mL of 50 mg/mL Kanamycin (Kana) Stock Solution:

1. Weigh 500 mg of kanamycin sulfate.
2. Add 10 mL of sterile distilled water and mix until fully dissolved.
3. Filter the solution through a 0.22 μm sterile filter to remove potential contaminants.
4. Dispense into sterile microcentrifuge tubes and store at -20 ℃.

Procedure:

1. All operations are performed in a sterile area.
2. Inoculate the culture medium with the inoculum at an appropriate ratio: 1:100.
3. Incubate the culture tubes on a bacterial incubator at 37 ℃ for 12–16 hours, shaking at a speed of 180–220 rpm.

The reaction system is as follows:

Procedure:

95 ℃ for 1 minute. Cycle 30-35 times for: 95 ℃ for 30 seconds, 56 ℃ for 30 seconds, 72 ℃ for 4 minutes. 72 ℃ for 5 minutes. Then 12 ℃ until the PCR products are taken out.

Adjust the extension time appropriately for different sizes of amplification products.

For single-clone PCR, pick a single colony from the culture plate as the amplification template instead of the DNA template.

Materials:

1×TAE buffer, agarose powder.

Equipment:

Gel casting tray, gel comb, electronic balance, graduated cylinder, Erlenmeyer flask, micropipette.

Formula:

Procedure:

1. Calculation:

For this experiment, a small gel casting tray is used, requiring 30 mL of TAE buffer and 0.45 g of agarose powder.

2. Measurement:

Weigh approximately 0.45 g of agarose powder using an electronic balance. Measure 30 mL of TAE buffer using a graduated cylinder and micropipette.

3. Mixing:

Add the agarose powder and TAE buffer into an appropriately sized Erlenmeyer flask. Gently swirl the flask until the agarose powder partially dissolves. Then, place the flask in a heating device (microwave) and heat until all agarose particles are completely dissolved. After heating, the solution should appear clear.

4. Staining:

Add 2 µL of nucleic acid stain to the solution and gently swirl until the solution shows a light pink color.

5. Gel Casting:

Slowly pour the prepared solution (agarose solution with nucleic acid stain) into the gel casting tray to avoid bubble formation, which can interfere with observation. Let the solution sit undisturbed until it solidifies into a gel.

1. Add 1 μLof 6× loading buffer to each 5 μLof PCR product and centrifuge to mix.
2. Remove the comb from the agarose gel and place the gel in the electrophoresis chamber.
3. Add an appropriate amount of electrophoresis buffer to the electrophoresis chamber.
4. Load the mixture into the wells of the gel and add DNA markers to the wells at both ends of the sample.
5. Turn on the electrophoresis instrument and set the electrophoresis parameters: 120 V, 20 minutes.
6. Observe the results under a blue light transilluminator or gel imaging system, such as the Gel Doc™ XR+.

Step 1: Scientists took 3 mL of the fifteen-hour cultured bacterial liquid and added it to a centrifuge tube. Then, it is centrifuged at 10,000 rpm (11,500×g) for 1 minute. The culture medium was then discarded as much as possible.

Step 2: Scientists then added 250 μLof Buffer P1that contains RNase A, to the centrifuge tube with the bacterial pellet, and mixed well with a vortex oscillator. This process resuspended the bacteria.

Step 3: Then, 250 μLof Buffer P2 is added to the centrifuge tube and is mixed with gentle inversion 8-10 times to lyse the bacteria fully.

Step 4: Then, 350 μLof Buffer P3 is added to the centrifuge tube, and is mixed with gentle inversion 8-10 times to neutralize Buffer P2 thoroughly. At this point, there should be white flocculent precipitation. The tube is then centrifuged at 12,000 rpm (13,400×g) for 10 minutes.

Step 5: Then, Scientists place the FastPure DNA Mini Columns in a 2 mL collection tube. The supernatant from the previous step is then transferred to the adsorption column with a pipette, and later centrifuged at 12,000 rpm (13,400×g) for 45 seconds. The waste liquid in the collection tube is then discarded. This process is meant for column loading and plasmid binding.

Step 6: Scientists then added 500 μLof Buffer PW1 to the adsorption column, which was centrifuged at 12,000 rpm (13,400×g) for 45 seconds. The waste liquid is then discarded. This process is to wash off the protein.

Step 7: 600 μLof Buffer PW2 diluted with anhydrous ethanol is then added to the adsorption column. Centrifuged at 12,000 rpm (13,400×g) for 45 seconds. The waste liquid is then discarded.

Step 8: The process above is repeated twice, washing off the salt ions.

Step 9: The adsorption column is then placed back in the collection tube and centrifuged at 12,000 rpm (13,400×g) for 1 minute to dry the adsorption column, removing the rest of the washing solution in the adsorption column.

Step 10: The adsorption column is then placed in a new sterile 1.5 mL centrifuge tube. Then, 100 μLof Elution Buffer is added to the center of the membrane of the adsorption column. The column is then placed at room temperature for 2 minutes, and centrifuged at 12,000 rpm (13,400×g) for 1 minute to elute the DNA. This process washes off the protein.

Step 11: The adsorption column is then discarded, and the DNA product is stored at -20℃ to prevent DNA degradation.

The reaction system is as follows:

Procedure:

1. Complete the whole experiment on ice.
2. Incubate the reaction mixtures at 37 ℃ for 10 minutes to complete the digestion and linearize the plasmid.

Process:

1. Transfer 50 μLenzyme-digested product and 100 μLPCR product into two different adsorption columns respectively, and then 12,000 rpm (13,800 ×g) centrifuge 40 seconds. DNA is adsorbed on the membrane of the adsorption column.
2. Discard the filtrate and place the adsorption column in the collection tube. Add 300 μLof Buffer GDP to the adsorption column. Let it stand for 1 minute, and then 12,000 rpm (13,800 ×g) centrifuge for 40 seconds.
3. Discard the filtrate and place the adsorption column in the collection tube. Add 700 μLBuffer GW (anhydrous ethanol has been added) to the adsorption column. Cover the lid, invert the collection tube 3 times to help thoroughly rinse off the salt adhering to the pipe wall, and then 12,000 rpm (13,800 ×g) centrifuge 40 seconds.
4. Repeat step 3 to make sure the salt is completely removed.
5. Discard the filtrate and place the adsorption column in the collection tube. 12,000 rpm (13,800 ×g) centrifuge for 2 minutes.
6. Place the adsorption column in 1.5 mL sterilized centrifuge tubes. Add 30 μLddH₂O into the middle of the adsorption column, place it for 2 minutes, and then 12,000 rpm (13,800 ×g) centrifuge 1 minute. Discard the adsorption column, detect the concentration of the products, and store the DNA at -20℃.

The reaction system is as follows:

Procedure:

1. Assemble the reaction system on ice.
2. Gently mix by pipetting up and down (avoid vortexing), then briefly centrifuge to collect the reaction mixture at the bottom of the tube.
3. Incubate at 50 ℃ for 15 minutes; then reduce the temperature to 4 ℃ or immediately place on ice for cooling.

Materials:

Procedure:

1. Thaw competent cells on ice.
2. Add 10 μL recombinant product to 100 μL competent cells, mix gently by flicking the tube (do not vortex), and incubate on ice for 30 minutes.
3. Heat-shock at 42 ℃ water bath for 45 seconds.
4. Place immediately on ice for 1 minute.
5. Add 890 μLLB medium (without antibiotics) and incubate at 37 ℃ for 60 minutes.
6. Spread 100 μL bacterial culture via the spread plate method.
7. Incubate at 37 ℃ for 12–16 hours.

Procedure:

1. Prepare a solid culture medium and sterilize it.
2. Allow the culture medium to cool to 60 ℃, then pour it into sterile agar plates.
3. Once the medium in the plates has solidified, use a pipette to add 50–100 μL of the culture to the center of the plate.
4. Use a spreader to evenly spread the bacteria across the surface of the medium.
5. Invert the plates and incubate at 37 ℃ for 16–24 hours.
6. Observe and record the growth status of the bacteria.

Prepare 200 mL of glycerin with a concentration of 40%: 80 mL glycerol plus 120 mL of ddH₂O, then high-temperature and high-pressure sterilization (121 ℃, 20 min).

Materials: bacterial culture.

Reagent: 40% glycerol.

Procedure:

1. Add 500 μL of bacterial culture and 500 μL of 40% glycerol to a sterile centrifuge tube.
2. Mix the bacterial culture and glycerol thoroughly.
3. Store it at -80 ℃.

Procedure:

1. Using a sterile pipette tip, pick a single bacterial colony.
2. Dip the tip into a tube containing 3 mL LB medium and vigorously vortex or flick the tube to dislodge the bacteria.
3. Place the inoculated tube in a 37 ℃ shaking incubator for 12–16 hours.

Materials: bacterial culture, microplate reader, 96-well microplate, culture medium

Protocol:

1. Prepare the microplate reader:

Turn on the microplate reader and set it to measure absorbance at 600 nm (OD600). Ensure that the instrument is properly calibrated.

2. Blank the microplate reader:

Add PBS or the same culture medium used for bacterial growth into one well of the microplate (typically 200 µL). This will act as the blank.

3. Prepare bacterial culture:

Shake or vortex the bacterial culture gently to ensure it is homogenous.

4. Add bacterial culture to the microplate:

Pipette an appropriate volume (typically 200 µL) of bacterial culture into the wells of the microplate.

5. Measure the OD600:

Place the microplate into the microplate reader and measure the absorbance at 600 nm (OD600). Record the values for each well.

6. Check bacterial growth stage:

The OD600 value correlates with bacterial cell concentration. Typically:

OD600 ~0.1-0.3: Log phase (rapid growth)

OD600 ~0.5-1.0: Mid-log phase

OD600 >1.0: Stationary or early stationary phase

Procedure

1. Expand the bacterial culture to an OD₆₀₀ of 0.6.
2. Add IPTG to the bacterial culture to achieve the appropriate final concentration.

If investigating the optimal IPTG concentration, set six different concentrations, such as 0 mM, 0.2 mM, 0.5 mM, 0.8 mM, 1 mM, and 2 mM, corresponding to different sample numbers.

The required IPTG volume is calculated using the dilution formula: n = c₁ × v₁ = c₂ × v₂.

For example, for sample 37-2, the final IPTG concentration is 0.2 mM, with a volume of 20 mL (v₁ = 20 × 10³ μL, c₁ = 0.2 mM), and the IPTG stock solution concentration is 0.5 M (c₂ = 0.5 × 10³ mM), the calculated volume (v₂) is: v₂ = (0.2 × 20 × 10³) / (0.5 × 10³) = 8 μL. Therefore, add 8 μL of IPTG to 20 mL of LB medium.

1. Incubate all samples at 37 ℃ with shaking at 220 rpm for 3 hours, or at 16 ℃ with shaking at 220 rpm for 20 hours.

Materials:

Procedure:

1. Bacterial lysis
1. Add 5 mL of 1×PBS buffer to the bacterial sedimentation and fully resuspend the bacteria.
2. Add 40 μL of lysozyme to a final concentration of 1 mg/ mL, mix well, and place on ice for 10 min.
3. Sonicate the bacteria on ice. Set the sonication power to 50%, turn on for 3 s and turn off for 3 s, and process for 5 min.
4. If the sonicated lysate is very viscous, repeatedly aspirate with a fine-needle syringe to shear the viscous genomic DNA. (optional)
2. Collect bacterial lysate

Centrifuge at 4 ℃, 5000 rpm for 10 min, collect the supernatant of the bacterial lysate, and place it on ice.

3. Equilibrate protein purification resin:

Take 1 mL of well-mixed 50% Beyogold His-tag Purification Resin, centrifuge at 4 ℃ (1000 ×g, 10 s), and discard the storage solution. Add 0.5 mL of non-denaturing lysis buffer to equilibrate the gel resin, centrifuge, discard the liquid, and equilibrate 1-2 more times, discarding the liquid each time.

4. Purify the protein
1. Mix 5 mL (all) of the bacterial lysate supernatant with the equilibrated Resin, and shake slowly at 4 ℃ for 60 min.
2. Load the mixture of bacterial lysate and Resin into an empty affinity chromatography column.
3. Open the cap at the bottom of the purification column, allow the liquid to flow out under gravity, and collect about 20 µL of the flow-through for analysis.
4. Wash the column 5 times, adding 0.5-1 mL of non-denaturing washing buffer each time.
5. Elute the target protein 6-10 times, using 0.5 mL of non-denaturing elution buffer each time.

The reaction system is as follows:

Procedure:

The RPA reaction tube contains white powder, which is a recombinase associated with the RPA reaction. The proportions of each component are based on the reaction system. Please note that 2 μL of magnesium acetate should be added to the tube cap.

After adding all components to the RPA reaction tube containing the powder according to the reaction system, close the tube cap and mix thoroughly. After briefly centrifuging, incubate the mixture at 42 ℃ for 20 minutes.

The reaction system is as follows:

Procedure:

The reaction mixture is incubated at 40 ℃ for 5–60 minutes, followed by inactivation at 85 ℃ for 10 minutes. The reaction product is cooled to room temperature before the results are analyzed.

For fluorescence detection systems, FAM-ssDNA-BHQ1 was used.

For strip testing, FAM-ssDNA-Biotin was substituted for ssDNA in the Cas12a cleavage reaction, compared to the fluorescent detection method.

Procedure:

1. Add 180 µL of ddH₂O to each Cas12a cleavage product.
2. Transfer the entire 200 µL solution into the wells of a non-transparent 96-well enzyme reaction plate.
3. Perform fluorescence measurement using a microplate reader.

Excitation wavelength: 492 nm,

Emission wavelength: 522 nm.

5 μL of Cas12a cleavage products were placed on the sample pad of the test strip. The test strip was then placed in 70 μL of 1× PBS buffer (containing 0.01% Tween-20) for chromatography. The appearance of a T line was observed within 2 min to determine the result.

1. Extraction of plasmid

Procedure:

1. Pick single colonies from transformation plates and inoculate them in liquid culture.
2. Inoculate the gum tip into 3 mL liquid culture medium in a tube. Place it on a shaker for cultivation for 12–16 hours (220 rpm).
3. Extract plasmid pET-28a-c .520G, pET-28a-c .520G>A, pET-28a-c.6745C, pET-28a-c.6745C>T from their bacteria solution.
2. Dilution of plasmid

Procedure:

Dilute the plasmid to obtain gradient samples with concentrations of 10⁸、10⁷、10⁶、10⁵、10⁴、10³、10²、10¹、10⁰ copies/Μl.

3. Preparation of random samples

Procedure:

For the two target sites, c.520G>A and c.6745C>T, standard plasmids containing both mutated and non-mutated sequences at varying concentrations (≥104 copies/µL) were randomly mixed. A total of 12 random samples were prepared.

Procedure:

1. Fluorescence detection system
1. RPA amplification
2. Protein degradation in RPA products
3. Agarose gel electrophoresis of RPA products
4. Cas12a cleavage of RPA products
5. Fluorescent detection of diluted Cas12a cleavage products

These experiments have been concluded before. For detailed steps, refer to previous experiments.

2. Test strip detection system
1. RPA amplification
2. Protein degradation in RPA products
3. Agarose gel electrophoresis of RPA products
4. Cas12a cleavage of RPA products

For strip testing, FAM-ssDNA-Biotin was substituted for ssDNA in the Cas12a cleavage reaction, compared to the fluorescent detection method.

5. Test strip analysis of Cas12a cleavage products

5 μL of Cas12a cleavage products were placed on the sample pad of the test strip. The test strip was then placed in 70 μL of 1× PBS buffer (containing 0.01% Tween-20) for chromatography. The appearance of a T line was observed within 2 min to determine the result.

These experiments have been concluded before. For detailed steps, refer to previous experiments.