Put the PCR tube into the PCR Cycler, and adjust the temperature at the different stages of the
reaction (denaturation, annealing, extension/elongation) and time (about 2 hours in total).
Gel Electrophoresis
Weigh 0.30g of agarose.
Add 30ml of 1x TAE solution.
Heat in a microwave and boil three times until the agarose is completely dissolved.
Mix well and let it cool to 50-60°C.
Add 3µl of nucleic acid dye.
Pour the mixture into a mold, insert the comb, and let it stand until it solidifies.
Mix 2µl of DNA with 0.4µl of 6x loading buffer.
Perform electrophoresis at 120v for 20 minutes.
Homologous Recombination
Plasmid template: Prepare 2.5 microliters each.
Since it is a 10-microliter reaction system, add 5 microliters of homologous recombination seamless cloning
enzyme.
Incubate in a PCR thermocycler at a constant temperature of 50°C for 20 minutes.
After the reaction is complete, place the centrifuge tube on ice and cool for a few seconds.
Bacterial Transformation
Add 10 microliters of homologous recombination product to 100 microliters of competent cells.
Thaw the competent cells on ice and add the DNA to the competent cells. Gently tap the tube to mix, and
incubate on ice for 30 minutes.
Heat shock at 42°C for 70 seconds, followed by incubation on ice for 5 minutes.
Add 500 μl of LB medium without antibiotics and shake the cells at 37°C for 1 hour to allow for recovery.
After centrifugation at 3000 rpm for 3 minutes, discard the supernatant, resuspend the cells, and plate them
on a solid medium containing 50 μg/ml kanamycin. Invert the plate and incubate overnight at 37°C in a
constant temperature incubator.
Plasmid Isolation
Column equilibration: Add 500 µl of Buffer BL to a centrifugal adsorption column, let it stand for 1 minute,
centrifuge at 12,000 rpm at room temperature for 1 minute, discard the waste from the collection tube, and
place the centrifugal adsorption column back into the collection tube. (Please use adsorption columns
prepared on the same day).
Bacterial collection: Take 1-5 ml of overnight cultured bacterial liquid (37°C, 12-16 hours) and add it to a
centrifuge tube (prepared by yourself). Centrifuge at 10,000 rpm at room temperature for 1 minute, and
completely remove the supernatant.
Resuspension: Add 250 µl of Buffer S1 containing RNaseA, and thoroughly mix by vortexing or gently pipetting
to ensure the bacteria are fully dispersed and suspended.
Lysis: Add 250 µl of Buffer S2, and gently invert the tube 5 times to mix, allowing the bacteria to lyse
completely until the solution becomes translucent.
Note: Avoid vigorous shaking to prevent genomic DNA from being fragmented for more than 5 minutes.
Neutralization: Add 350 µl of Buffer S3, gently invert the tube 5 times to mix thoroughly, avoiding vigorous
shaking, and centrifuge at 12,000 rpm at room temperature for 2 minutes.
DNA binding: Carefully transfer the supernatant to the centrifugal adsorption column inserted into the
collection tube, centrifuge at 12,000 rpm at room temperature for 30 seconds, discard the waste from the
collection tube, and reinsert the centrifugal adsorption column into the collection tube.
Wash: Add 600 µl of Buffer WB to the centrifugal adsorption column, let it stand for one minute, and
centrifuge at 12,000 rpm for 30 seconds.
Empty spin: Place the adsorption column back onto the collection tube and centrifuge at 12,000 rpm for 1
minute.
Elution: Carefully remove the centrifugal adsorption column and transfer it to a 1.5 ml centrifuge tube. Add
50 µl of preheated Buffer EB to the center of the silica membrane, let it sit at room temperature for 1
minute, then centrifuge at 12,000 rpm for 1 minute to collect the plasmid DNA.
Repeat the step 9 by reapplying the DNA liquid back into the adsorption column, letting it sit at room
temperature for 3 minutes, then centrifuging at 12,000 rpm for 1 minute to collect the plasmid DNA.