1. Required substances: 5g peptone, 2.5g yeast extract, 5g NaCl, 500ml deionized water to prepare LB culture medium
2. Put the culture medium into a shaker to mix up
3. After shaking, add 10g of agarose (1g of agarose per 100ml)
4. Use of sterilizer: first take out the sterilization frame, then add enough water to the pot, so that the water surface is slightly below the triangular shelf
5. Put back the sterilization frame and fill it with the items that need to be sterilized. Be careful not to pack too much so as to prevent the steam flow and affects the sterilization effect. Do not contact the wall of the triangular flask and the mouth of the test tube to avoid condensation from wetting the paper at the mouth of the package and penetrating the cotton plug
6. Add the lid, then tighten the two bolts in a symmetrical manner, so that the bolts are in the same situation (or the rotating disk on the sterilization pot is tightened), and do not make air leakage
7. Set the sterilization temperature to 121℃ and time to 25 minutes, and open the exhaust valve at the same time, turn on the power to make the water boil to exclude the cold air in the pot. After the cold air is completely exhausted (at about 109°C), close the exhaust valve and let the temperature inside the pot rise gradually with the increase of steam pressure. When the pressure in the pot rises to the desired pressure, the heat source is automatically controlled to maintain the pressure for the desired time
8. When the required time for sterilization is up, cut off the power supply and let the temperature inside the sterilizer drop naturally. When the pressure in the pressure gauge drops to 0, open the exhaust valve, loosen the bolt, open the lid and take out the sterilized items. If you open the exhaust valve when the pressure does not drop to 0, the pressure inside the pot would drop suddenly, causing the culture medium inside the container to rush out of the flask mouth or test tube mouth due to the imbalance of internal and external pressure, resulting in contamination by cotton plugs staining the culture medium
9. After completing sterilization, wait for the medium temperature to be about 50℃, then move it into the Petri dish for cooling

1. Take an LB solid medium plate (non-antibodies) and place it in a 37°C incubator to preheat
2. Take a tube of Top10 competent cells, burn the inoculation loop to sterilize, and cool it (lean against the inner wall a few times)
3. Then dip it in Top10 bacterial solution and line on the preheated plate (draw three to five parallel lines quickly with the inoculation loop dipped in the strain, cauterize the inoculation loop again, cool it, lean against the blank medium a few times, start drawing lines from the end of the first area, repeat the above steps in the third area to avoid connecting one and three areas)
4. The plate is inverted at 37℃ and incubated overnight. When the colony grows up, it can be stored at 4℃
5. Inoculate a single colony with a pipette into LB liquid medium (without antibiotics) and incubate overnight at 37°C, 200 rpm
6. Transfer 1ml of the cultured medium into 100 mL fresh LB-liquid medium (without antibiotics)
7. 37°C, 200 rpm, shake until OD600 is 0.3-0.5 (competent cells are suitable for about 0.4)
8. Place the conical flask directly in the ice bath for 15 min
9. Transfer 100 mL of the bacterial solution to two 50 mL centrifuge tubes (pre-cooled in the ice bath)
10. Centrifuge at 4500 rpm, 4°C, for 8 min (cool down the centrifuge in advance)
11. Discard the supernatant and add 2 mL of sterile water to each tube and gently resuspend the cells (dispense an appropriate amount of sterile water from the ultra-clean table and pre-chill on ice in advance)
12. Centrifuge at 4500 rpm, 4°C, for 8 min
13. Discard supernatant, centrifuge briefly, and aspirate supernatant
14. Add 5 mL of (0.05 M CaCl2 + 15% glycerol) premix to each tube, blow gently, resuspend the organism and bathe on ice for 45 min. (operate on ice, use a sterilized gun tip on the ultra-clean table)
15. Centrifuge, 4500 rpm, 4°C, 8 min
16. Discard the supernatant and aspirate the liquid.
17. Add 2 mL (0.05 M CaCl2 +15% glycerol) premix into tube ① first, resuspend the bacteria, then transfer to tube ②, then wash tube ① with 1 mL premix, transfer to tube ②, and mix 3 mL of bacteria in tube ②, mix well
18. Dispensation (100 μL per tube), and label the tube with the strain name and freeze at -80℃

1. Dissolve agarose and prepare the gel
2. Add an appropriate amount of electrophoresis buffer and note that the volume should not exceed one-third of the conical flask.
3. Add ethidium bromide to a concentration of 0.5 mg/ml
4. First cool the dissolved agarose gel solution in a 65 ℃ water bath, and DNA nucleic dye
5. Put the comb into the glue to form a sample loading hole. Pour the dissolved agarose gel into the mold and then solidify at room temperature
6. After the agarose gel solidifies, remove the comb
7. Before starting electrophoresis, add the loading buffer to the DNA sample to be separated. Set the power supply to the specified voltage. Add enough electrophoresis buffer into the electrophoresis tank until it covers the surface of the gel. Plug the connector of the electrophoresis tank into the power supply and then turn on the power supply
8. Remove the electrophoresis tank cover and slowly and carefully add DNA samples. Cover the electrophoresis tank and check again whether the electrode is plugged into the correct socket of the power supply
9. Turn on the power and start electrophoresis until the dye reaches the appropriate position of the gel
10. Observe the isolated DNA fragments. First, take out the gel from the glue tank and irradiate it with the UV lamp
11. After electrophoresis, turn off the power and remove the electrophoresis tank cover. Take out the gel from the electrophoresis tank and drain the excess electrophoresis buffer on the surface. Put the glue tank on a paper towel and suck up the remaining electrophoresis buffer

1. Cut down the gel containing the target fragment and calculate the weight difference in the EP tube from adding the gel by weighing the tube before and after adding the gel
2. Add 600 μl buffer into the tube, place it in a 60 ℃ water bath for 10min, and mix it every 2 minutes to make the rubber block melt completely
3. Transfer the entire dissolved solution to the adsorbent column and place it at room temperature for 2 min
4. Centrifuge at 8000 rpm for 1 min at room temperature and discard the filtrate in the collection tube
5. Put the adsorption column into the same collection pipe and add 500 μ L wash solution, 8000rpm, centrifuged at room temperature for 1min
6. Repeat the 4th step
7. Remove the adsorption column, discard the filtrate, put the adsorption column back into the same collection tube, and centrifuge at room temperature at 12000 rpm for 1 minute
8. Put the adsorption column into a new 1.5 ml centrifuge tube, add 40ml elution buffer in the center of the column, and place for 2 minutes at room temperature or 37 ℃
9. Centrifuge at 12000rpm for 1 min at room temperature and the liquid in the centrifuge tube is the recovered product

1. Use a pipette to add reagents in a PCR tube and mix up (which contains enzymes required for PCR; dNTPs; ddH2O; and Buffer). Next, add the primers into the tube. Afterward, divide the prepared mixture into 2 PCR tubes.

2. Put the PCR tube into the PCR machine. Close the lid and set the program.

Steps	Temperature	Time	Number of cycles
Initial denaturation	94 ℃	5 min	/
Denaturation	94 ℃	30 sec	/
Primer annealing	55 ℃	45 sec	33
Extension	72 ℃	2 min	33
Final extension	72 ℃	5 min	/

1. Inoculate the bacteria and culture overnight, and centrifuge the cultured medium at 12000rpm for 1 min. Discard the supernatant
2. Add 500μl of the balance solution to the adsorption column, centrifuge the discard the filtrate
3. Add 250μl of Buffer SP1 from the Diamond Mini Plasmid Kit to the pellet cells. Add 250μl Buffer SP2 and 350μl Buffer SP3 and mix well.
4. Centrifuge at 12000rpm for 10 minutes, add the supernatant to the adsorption column, and centrifuge at 8000rpm for 30s.
5. Add 500 mL of wash solution, centrifuge at 9000×g for 30 seconds, and pour off the liquid in the tube (repeat once)
6. Centrifuge the empty adsorption column at 9000 ×g for 1 minute
7. Put the adsorption column into a new 1.5mL centrifuge tube, add 100 mL of Elution Buffer to the adsorption membrane, stand at room temperature for 1 minute, and centrifuge for 1 minute.

1. In the process of PCR amplification, the terminal homologous sequence of the linearized cloning vector was introduced at the TSD end and TIR end
2. The assembly enzyme and PCR-amplified products are mixed together so that the insert can be positioned on the linearized vector. Assembly enzyme catalyzes a homologous recombination reaction to form a recombinant vector
3. Transform the recombinant plasmids into competent cells and coat on the LB solid medium plate

1. Inoculate the transformed strain, incubate at 37 ℃for 12 ~ 16 hours
2. Add 1.5ml bacterial solution into EP centrifuge tube, centrifuge 12000g for 30 seconds, and discard the supernatant
3. Add 100μl of cooled solution I, resuspend bacterial cells on the vortex oscillator to disperse the cells as much as possible
4. Add 200μl of newly prepared solution II, quickly reverse the centrifuge tube to mix the contents, and place it on ice for 3-5min (NaOH in solution II can cleave cells, denature DNA and denature proteins to form a cross-linked network structure)
5. Add 150μl of solution III, reverse it 6-7 times after capping, mix it evenly, and place it on ice for 2 ~ 3min (Solution III is a low pH potassium acetate buffer that neutralizes NaOH to renature partially denatured closed-loop plasmids, while bacterial chromosomal DNA cannot be renatured correctly)
6. Centrifuge 12000 g for 6 min, and transfer the supernatant into another clean EP tube
7. Add 2 times the volume of supernatant (about 1ml) of absolute ethanol, shake and mix well, and place at room temperature for 2min
8. Centrifuge at 12000g for 10min, the supernatant was discarded, and then washed once with 70% ethanol. Centrifuge at 12000g for 1min, discard the liquid
9. Add 40ml of sterile distilled water extract containing 20 mg/ml RNase A, place it at room temperature until the plasmid is completely dissolved (about 8min), store it at - 20 ℃ or directly use it for enzyme digestion

1. Mix the target gene product with casposase protein in equal proportion. React in a metal bath at 37°C for 1h
2. Add 4uL PK enzyme per tube (PK/0.5M EDTA v/v=1:1)
3. 37℃ metal bath digestion for 30min
4. 95℃ 10min inactivation 100uL/tube isopropanol precipitation 1h
5. Recovery of integration product 100uL DH10b (electrotransfer)/sample
6. Add 1mL SOC medium Recovery for 1h
7. Centrifuge at 4000rpm for 8min, discard 850uL supernatant
8. Blow the remaining well, coat all, and pick monoclonal
9. Plasmid extraction