1.Thaw the ingredients on ice and add the specified volumes for each group as needed (see Table 1).
2.Gently centrifuge the mixture and mix well.
3.Transfer the PCR tube to the PCR instrument, set the parameters, and start the polymerase chain reaction.
4.Store the reagents at -20 °C for long-term storage.
Table 1 Reaction systems
| Components | Volume |
| ddH2O | Up to 50µL |
| 2×Phanta SE Buffer | 25µL |
| Primer F (10 μM ) | 2µL |
| Primer R (10 μM ) | 2µL |
| Phanta SE Super-Fidelity DNA Polymerase | 1µL |
| Template DNA | xµL |
Table 2 Standardized reaction procedures
| Cycle steps | Temperature | Time | Cycles |
| Predegeneration | 98°C | 30 sec | |
| Transgender | 98°C | 10 sec | |
| Annealing | Tm | 5 sec | 28-35cycles |
| Extension | 72°C | 5 ~ 10 sec/kb | |
| Completely Stretch | 72°C | 1 min |
1.Mix agarose and 1× TAE buffer at a ratio of 1 g:100 mL as desired, and pour the mixture into a 250 mL conical flask. Place the flask in the microwave and heat until the agarose is completely dissolved.
2.Add the staining solution at a ratio of 1 µL of 4S Green Plus to 20 mL of TAE buffer.
3.Pour the solution into the tray and insert the comb with the appropriate number of wells.
4.Allow the gel to cool and solidify, then carefully remove the comb. Place the gel into the electrophoresis apparatus, ensuring there is enough 1× TAE buffer.
5.Add 10× DNA Loading Buffer to the samples, mix well, and transfer the mixture to the wells of the gel using a pipette.
6.Turn on the power and run the electrophoresis for 30 minutes at 105 V.
The corresponding bands were quickly excised under UV light for gel recovery using the Novizen Product Purification Kit.
1.After completing DNA electrophoresis, carefully cut out the gel containing the target DNA fragment under UV light. It is advisable to use a paper towel to absorb any liquid on the gel's surface before cutting, ensuring that excess gel is removed. Weigh the gel (excluding the weight of the empty tube); 100 mg of gel corresponds to a volume of 100 µL. Add an equal volume of GDP buffer. Incubate in a water bath at 50-55℃ for 7-10 minutes, adjusting the time based on the gel size to ensure complete dissolution. During incubation, invert and mix the solution twice to accelerate the process.
2.Briefly centrifuge to collect any droplets from the tube walls. Place the FastPure DNAmini Columns-G adsorption column into a 2 mL collection tube and transfer≤700 µL of the solubilized solution to the adsorption column. Centrifuge at 12,000 rpm (13,800 × g) for 30-60 seconds.
3.Discard the filtrate and place the adsorption column back into the collection tube. Add 300 µL of Buffer GDP to the adsorption column and let it stand for 1 minute. Centrifuge again at 12,000 rpm (13,800 × g) for 30-60 seconds.
4.Discard the filtrate and place the adsorption column in the collection tube. Add 700 µL of Buffer GW (with anhydrous ethanol added) to the adsorption column. Centrifuge at 12,000 rpm (13,800 × g) for 30-60 seconds. (To ensure complete removal of salts adhering to the tube walls, it is helpful to add Buffer GW around the column wall or mix by inverting the cover and mixing 2-3 times.)
5.Repeat step 4.
6.Discard the filtrate and place the adsorption column in the recovery header. Centrifuge at 12,000 rpm (13,800 × g) for 2 minutes. Place the adsorption column into a 1.5 mL sterile centrifuge tube, add 20–30 µL of Elution Buffer to the center of the column, and let it stand for 2 minutes.
7.Centrifuge at 12,000 rpm (13,800 × g) for 1 minute. Discard the adsorption column and store the DNA at -20℃. For optimal yield, it is recommended to add the eluted solution back to the centrifugal adsorption column and repeat this elution step. When recovering fragments larger than 3 kb, preheating the Elution Buffer to 55℃ can improve recovery efficiency.
The process uses Novozan ClonExpress Ultra One Step Cloning Kit V2 kit:
1. Calculate the amount of DNA used for recombination reaction:
◇For single fragment homologous recombination reactions.
The optimal use amount of cloningVector=[0.02×base logarithm of cloningVector] ng (0.03 pmol)
The optimal amount of inserted fragments=[0.04×base pairs of inserted fragments] ng (0.06 pmol)
◇For multifragment homologous recombination reactions.
The optimal use amount of cloningVector=[0.02×base logarithm of cloningVector] ng (0.03pmol)
The optimal dosage of each fragment=[0.02×base logarithm of each fragment] ng (0.03pmol)
Prepare the following reaction system under ice (10µL reaction system).
Table 3 Cloning reaction system
| Reagents | Volume |
| Linearized carriers | xµL |
| N Inserting clips | Y1 - YnµL |
| 2×CE Mix | 5µL |
| ddH2O | Up to 10µL |
1. Mix gently by pipetting (do not shake) and collect the reaction solution to the bottom of the tube by centrifuging briefly.
2. Recombination reaction.
Single fragment reconstitution reaction, 50℃, 5min; reduce to 4℃ or cool immediately on ice.
2 ~ 3 fragment recombination reaction, 50℃, 15min; lower to 4℃ or cool immediately on ice.
The recombinant products can be stored at -20℃ for one week and can be thawed for transformation.
1.Remove the competent cells and plasmids from the -80 ℃ and -20 ℃ refrigerators, respectively, and thaw them on ice.
2.In a tube, add 100 µL of thawed competent cells and 10 µL of plasmid. Gently mix the solution and incubate on ice for 30 minutes.
3.Heat shock the mixture at 42℃ for 90 seconds, then quickly return it to ice for 2-3 minutes without shaking.
4.Add 700 µL of antibiotic-free LB liquid culture medium to the cells containing the plasmids and incubate in a shaker at 37 ℃ for 1 hour.
5.Take 100 µL of the bacterial solution and spread it on LB agar plates containing the appropriate antibiotics.
6.Invert the petri dishes and incubate at 37℃ overnight in a constant-temperature environment.
Table 4 Colony PCR reaction system
| Composition | Volume |
| 2×Rapid Taq Master Mix | 5µL |
| Upstream primer (100uM) | 0.4µL |
| Downstream primer (100uM) | 0.4µL |
| Colonies | Not counting |
| ddH2O | Up to 10µL |
1.Prepare the PCR reaction solution according to the specified system.
2.Select multiple single colonies from the transformed petri dishes incubated overnight and transfer them into the reaction system using a sterile tip.
3.Complete the reaction and verify it using nucleic acid gel electrophoresis. If fragments of the appropriate size are present, streak the colony plates and incubate them before transferring to liquid medium. Store the strain in a 1:1 solution of glycerol and bacteria, and send the remaining samples for sequencing.
1.Take 50 µL of the preserved bacterial solution and incubate it in a 5 mL shaking tube containing LB liquid medium (with the appropriate antibiotic) for 12 hours at 37 ℃ and 200 rpm.
2.Inoculate 50 mL of LB liquid medium with 2% of the activated bacterial solution and culture it at 37 ℃ and 200 rpm until it reaches an OD₆₀₀ of 0.6.
3.Add IPTG to achieve a final concentration of 0.5 mM, then induce the culture for 12-16 hours at 20 ℃ and 200 rpm.
1.Pour 30 mL of the bacterial solution into a 50 mL centrifuge tube, balance it, and centrifuge at 8000 rpm for 5 minutes. Discard the supernatant and add more bacterial solution to the tube. After balancing with RO water, centrifuge again and discard the supernatant. Finally, add 5 mL of PBS for resuspension, centrifuge, and discard the supernatant.
2.Once all bacteria are collected, invert the centrifuge tube on paper for a short time and wipe the nozzle dry. Add 2 mL of PBS to resuspend the bacterial pellet and transfer it into the corresponding 5 mL centrifuge tube. Then, add 1 mL of PBS to clean the centrifuge tube and pour this into the 5 mL tube.
3.Place the centrifuge tube in a floating plate and into a box containing an ice-water mixture. Adjust the upper and lower positions, adding ice as needed to maintain a low temperature. Turn on the ultrasound device, ensuring the power is around 300 W at the start. Crush the solution for 3 seconds, followed by a 5 second interval, for a total duration of 10 minutes.
4.Incubate the crushed bacterial solution at 4 ℃ and 5000 rpm for 20 minutes.
5.Separate the supernatant from the pellet for later use. Resuspend the pellet in 2 mL of PBS and take a sample. The supernatant can be purified and stored in a refrigerator at -20℃.
1.Prepare the nickel column (stored in -20℃ alcohol) and wash it with 5 mL of Buffer A, followed by 3 mL of Buffer B, and then 5 mL of Buffer A after discharging the alcohol.
2.Add the crude enzyme solution to the column and wash twice with 5 mL of Buffer A to remove any impurities. Then, add 3 mL of Buffer B and collect the eluent in a beaker for labeling to obtain the target protein.
3.Wash the column with 3 mL of Buffer B and then 5 mL of Buffer A, and seal the column with 20% alcohol.
4.Pour the crude enzyme solution from the beaker into an ultrafiltration tube (sealed with water on top) and centrifuge at -4℃ and 6500 rpm for 30 minutes.
5.After that, add 2 mL of water, centrifuge at 6500 rpm for 30 minutes.
6.Finish with 2 mL of water, centrifuge at 6500 rpm for 15 minutes. If the volume of the final concentrated pure enzyme solution exceeds 500-700 µL, centrifuge for an additional 5-15 minutes to increase protein concentration.
7.Store the final concentrated pure enzyme solution in a refrigerator at -20℃.
According to the size of the target protein, different concentrations of separation gels were prepared, with the concentrated gel set at 5%. The specific steps are as follows:
1.Protein Gel Preparation: Assemble the mold and ensure it is leak-proof. Prepare the appropriate concentration of separating gel based on the instructions from the SDS-PAGE gel preparation kit (Beijing Soleberg Technology Co., Ltd.). Pour the separating gel into the glass plate up to two-thirds full and seal the remaining space with water. Once the gel has solidified, pour off the upper layer of water and dry the surface with filter paper. Immediately pour the 5% concentrated gel on top of the separating gel and insert the tooth comb. Allow the concentrated gel to solidify completely, then wrap it and store it in the refrigerator at 4℃ for later use.
2.Protein Sample Processing: Dilute 9 µL of protein to the desired concentration, then add 3 µL of 4× protein SDS-PAGE loading buffer. Heat the mixture at 100℃ for 10 minutes, cool it to room temperature, and centrifuge to collect the sample.
3.Electrophoresis Process: Mount the protein maker plate in the electrophoresis equipment and add freshly diluted 1× SDS-PAGE protein sample loading buffer. Remove the tooth comb and load 5 µL of the appropriate range of protein maker along with 20 µL of the protein sample. Perform electrophoresis at 120 V until the bromophenol blue reaches the bottom of the glass plate. Remove the protein gel and immerse it in a protein staining solution for about 40 minutes, then decolorize by gently shaking in a protein decolorizing solution.
FastPure® plasmidmini Kit (Vazyme) was used to rapid purification and extraction of plasmid DNA.
1. Take 5 ~ 15mL of overnight cultured bacterial liquid, centrifuge at 12000 rpm (13400×g) for 1min to collect the bacterial body, and try to suck out the residual supernatant.
2. Add 500µL Buffer P1 (please check whether RNase A Solution has been added first), and Vortex until the cell is completely suspended.
3. Immediately add 500µL Buffer P2, gently mix it upside down for 8 ~ 10 times, and place it at room temperature for 3min. (This step should be gently mixed, not violently shaken, to avoid genomic DNA contamination. At this time, the solution should gradually change from turbid and viscous blue to clear and viscous blue, indicating complete cracking. The time used should not exceed 5minutes to avoid damage to the plasmid. If it does not become clear, it may be due to excessive bacterial growth and incomplete lysis, and the bacterial volume should be reduced.)
4. Add 500µL Buffer N3, immediately and gently invert it up and down for 12 ~ 15 times until the blue color disappears completely. At this time, white flocculent precipitates should appear. Place it at room temperature for 10min, and centrifuge it at 12000 rpm (13400×g) for 10min. (After Buffer N3 is added, it should be immediately gently reversed and mixed to avoid local precipitation. At this time, the solution will change from blue to colorless, indicating complete neutralization; after centrifugation, the supernatant should be clear, and if there is a small white precipitation on the supernatant surface, it will not affect subsequent operations)
5. Transfer the supernatant (about 1.5mL) to a 5mL centrifuge tube (self prepared), add 0.3 times the volume of isopropyl alcohol (about 450µL), and mix it up and down for 10-15 times.
6. Column balance: Place FastPure DNAmini Columns III in 2mL Collection Tubes. Add 200µL Buffer QB to FastPure DNA Mini Columns III, and centrifuge at 12000 rpm (13400×g) for 1min. Then discard the filtrate, and reserve it.
7. Transfer the mixed solution in step 5 to the adsorption column, centrifuge at 12000 rpm (13400×g) for 1min, and discard the filtrate. To increase the elution efficiency of plasmid, the eluent can be preheated at 65℃. In order to increase the elution yield, the centrifugal solution can be eluted on the column twice.
8. Repeat step 7 until the mixture is all on the column.
9. Add 500µL Buffer PW1 to the adsorption column, centrifuge at 12000 rpm (13400×g) for 1min, and discard the filtrate.
10. Add 600µL Buffer PW2 (please check whether absolute ethanol has been added first) to the adsorption column, centrifuge at 12000 rpm (13400×g) for 1min, and discard the filtrate.
11. Repeat step 10.
12. Put the adsorption column into the recovery header and centrifuge it for 2min at 12000 rpm (13400×g) to dry the adsorption column.
13. Place the adsorption column in a new 1.5mL centrifuge tube (self prepared), and add 60 ~ 200µL Caution Buffer to the center of the membrane of the adsorption column. Let it stand at room temperature for 1min, centrifuge at 12000 rpm (13400×g) for 1min, and discard the adsorption column.
14. The extracted plasmid DNA is stored at -30~-15℃.
Select a single colony of Escherichia coli and inoculate it into LB liquid medium. After 20 hours of cultivation, dilute the culture to an OD₆₀₀ of 0.1. Incubate at 37 ℃ for 2 to 3 hours, then add IPTG to a final concentration of 0.2 mM. After 12 hours of induction, take 1.5 mL of the bacterial culture and centrifuge at 12,000 rpm for 2 minutes. Discard the supernatant and wash the cells with 1 mL of 1× PBS buffer. Prepare a microscope slide with an appropriate amount of cells and observe the fluorescence intensity of Bs2 under excitation light at 447 nm.
1.Plug in the power and turn on the power switch of the ELISA reader by pressing the switch on the back of the instrument, and also switch on the connection to the computer.
2.Open the software to access the main operating interface.
3.Select "Start Measurement," then choose the appropriate measurement project and the layout of the plate being used.
4.Click "OK," followed by the "Start" button.
5.The instrument will enter measurement mode: during this time, the operator should refrain from performing any other operations.
6.Once the measurement is complete, the test results will be displayed on the monitor. Select the option to save the measurement data.
7.Finally, turn off the power to the ELISA reader.
1.Instrument Inspection: Check the status of the HPLC equipment to ensure the proper functioning of the column, detector, pump, and other components.
2.Sample Preparation: Prepare the sample to be tested, ensuring it is suitable for HPLC analysis.
3.Sample Dissolution: Dissolve the sample in an appropriate solvent to achieve the desired concentration.
4.Column and Mobile Phase Selection: Choose the appropriate type and size of column for the analysis, and prepare a suitable mobile phase, including solvents and buffers, ensuring compatibility with both the selected column and the sample.
5.HPLC Parameter Setup: Set the HPLC parameters, adjusting the pump flow rate according to the column specifications and sample properties. Select and configure the detector as needed, and adjust the column temperature to optimize separation.
6.System Start-Up: Begin the HPLC system, ensuring a stable flow of the mobile phase through the column.
7.Data Recording: During operation, record the detector output data, including peak heights and retention times.
8.Data Analysis: Analyze the data based on the chromatograms, observing and identifying the peaks of different components; use the area of the peaks to calculate the relative concentration of each component.
9.System Shutdown: After completing the analysis, flush the system according to the instrument's requirements to prevent cross-contamination, and disconnect the power supply after flushing.