Plasmid construction

PCR instrument; Electrophoresis apparatus; Gel imager; Bechtop; Centrifuge; Sequencers or sequencing companies

Plasmid construction is the most commonly used experimental technique in molecular biology research. The principle relies on the action of restriction endonuclease, DNA ligase and other modifying enzymes. After proper cutting and modification of the target gene and DNA carrier, the two are joined together and then introduced into the host cell to achieve the correct expression of the target gene in the host cell^[1].

MCE has not independently verified the accuracy of these methods. They are for reference only.

1. The inserted gene fragment is amplified by PCR according to the instructions of the PCR reagent used and linked to the primer sequence, which contains the sequence overlapping with the plasmid restriction site and the target gene.

2. The plasmid skeleton was linearized by enzyme digestion according to the instructions for the restriction enzymes used.

3. Treat the linearized plasmid skeleton according to the instructions for alkaline phosphatase used.

4. Run PCR products and linearized skeleton in agarose gel to confirm size.

5. Recover purified DNA from gel according to the instructions of the gel recovery kit used.

6. Ligate the linearized plasmid skeleton and insert gene fragments according to the instructions of T4 ligase used.

7. According to the instructions of the competent cells used, the ligation product is transformed into the competent cells.

8. Colony PCR and electrophoresis were performed to screen clones with successfully inserted genes.

9. Sequencing to verify that the inserted gene is correct.

1. The selection of vector cloning sites should be performed in regions with no repetitive sequences and relatively uniform GC content. When the GC content in the upstream and downstream 20 bp region of the cloning site was within the range of 40% ~ 60%, the cloning efficiency would reach the maximum.

2. The optimal molar ratio of plasmid skeleton to inserted fragment was 1:2.

3. It is best to use enzymes from the same company in the same enzyme digestion system.