1. PCR
2. PCR Gel Electrophoresis
3. PCR Gel Purification
4. Double-enzyme digestion
5. T4 DNA ligase
6. DNA transformation to competent cells

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.

1) Since the DNA contains a negative electric charge, we placed the gel with wells toward the negative charge of the buffer tank, as the other side is positively charged. Keep in mind that the TAE buffer needs to fully cover the gel.
2) Added DNA loading buffer to the PCR product, according to its concentration.
3) After the electrophoresis, we put the gel under the ultra slim LED illuminator to see the DNA and cut out the gel with DNA fragments for the next step.

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 500µL B2 buffer to the gel, and water bath at 50℃ for 5-10 minutes to make the gel melt in the buffer
3) Move all the liquid into the absorption column with membrane, centrifuge for 30s, at 8000×g
4) Add 500µL Wash Solution, 9000×g centrifuge for 30s, repeat this step again
5) Centrifuge again for 2 minutes, 12000×g.
6) Put the absorption column into a clean 1.5mL centrifuge tube, add 30µL Elution Buffer, put it at room temperature for 2 minutes, and centrifuge for 1 minute to keep the DNA solution.
7) Measure the DNA concentration using the Nanodrop machine.

1) add 2 μL each of the restriction enzyme into one micro-centrifuge tube.
2) add 5μL buffer, 10μL ddH₂O, and 1μL DNA into each micro-centrifuge tube.
3) 37℃ incubation for 1h.
4) Agarose Gel Electrophoresis and gel extraction.

1) add 1 μL T4 DNA ligase, 1 μL T4 DNA ligase buffer, 1 μL DNA fragment, 1 μL plasmid vector, and 5 μL ddH₂O into the microcentrifuge tube.
2) 22 ℃ water bath for 30 minutes.

1) Put the chemically competent cells DH5α on ice for 1-2 minutes.
2) Mix the DH5α competent cells with precooled recombinant plasmids. Ice bath for 30 minutes.
3) Heat shock at 42°C for 1 minute and put on ice immediately for 2 minutes.
4) Add 700μL LB medium and incubate it at 37℃, 200rpm for 60 minutes.
5) Centrifuge 8000rpm 1 min, get rid of 650µL of supernatant.
6) Resuspend the E. coli and coat it on the solid medium.

1. Colony PCR
2. Plasmid Extraction

ystem:
1. We added 5µL ddH₂O .
2. Added 10µL Primer S. Mixer (which includes dNTP, DNA polymerase, and reaction buffer).
3. Added 3µL of DNA template.
4. Added 1µL each of forward and reverse primer.
program:
1) 95℃, 5min, pre-degenerate
2) 95℃, 10s, DNA degeneration
3) 58℃, 30s, annealing
4) 72℃, elongation
5) 72℃, 10min, fully elongate
6) 4℃, infinite time, preserve

We cultured E. coli that to do plasmid extraction
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.