Molecular Cloning

Q5 DNA polymerase PCR system(50µL ) 25µL 2µL

2µL

1µL

20µL

Enzyme digestion Connection

Enzyme digestion and connection system(10µL )

3µL

1µL

1µL

0.5µL

4.5µL

Connection system

Transformation

Take 50 µL of competent cells in a sterile Eppendorf tube.

Add 2~5 µL of the plasmid to be transformed into each tube, gently mix the plasmid and competent cells with a pipette tip, and ice bath for 25 minutes.

Heat shock the ice-bath mixture in a 42℃ water bath for 45s, do not shake the centrifuge tube.

Take out the ice bath and cool for 2 minutes.

Add 800 µL of non-resistant LB medium preheated to 37°C to each tube, shake gently at 37°C, 150r/min for 1h.

Centrifugally concentrate to 100μL, spread it on an LB plate containing antibiotics, and incubate inverted at 37°C for 12-16h.

Make a negative control at the same time (use distilled water instead of plasmid).

Colony PCR screening of positive clones

Add 25ul of sterile water to the 96-well plate, pick about 15-20 colonies from the successfully transformed DH5α bacterial plate with a pipette tip, and mix them evenly. (Make a corresponding mark on the picking plate).

Put the mixed bacteria solution into 10ul to 200ul PCR tube and mark it.

Take 0.1ul of bacterial solution in a 96-well plate and add it to the antibiotic-containing plate with a pipette tip and mark it.

After mixing, the bacteria solution is used for PCR, the system is as follows(20µL )

5µL

0.5µL

0.5µL

2µL

2µL

The procedure is as follows

Verification result of 1% agarose gel electrophoresis.