You understandThe process of plasmid construction!Is it?Plasmid construction is the most commonly used experimental technique in molecular biology research, which relies on restriction endonucleases to appropriately cleave and modify the target gene and vector DNA, and then use DNA ligase to link the two together and then introduce them into the host cell to achieve the correct expression of the target gene in the host cell.
Plasmid constructionofprocessIt can be divided into the following steps:
Plasmid extraction - amplification of the gene fragment of interest - enzymatic digestion of the plasmid and the fragment of interest - ligation - transformation - culture - identification.
1. Preparation of plasmid vectors.
Plasmid vectors can be prepared with either single or double digestion, and double digestion is generally recommended. In fact, there is only one purpose, to make the end of the carrier as specific as possible to prevent self-connection. In general, when the gene of interest is used for clonal expression, the selection of the enzyme cleavage site should be considered
1) The distance between the two enzyme cleavage sites on the plasmid should be 10bp, otherwise it will affect the recognition of the restriction enzyme to the cleavage site, which is not conducive to the double digestion of the empty vector
2) The gene fragment of interest does not contain the selected enzyme cleavage site
3) The vectors (eukaryotic, prokaryotic, yeast, insects) used in the experiment should contain the selected digestion site as much as possible
4) The two digestion sites are at least 3 bases apart, preferably not with the same tail enzyme (residues may be complementary);
5) It is best to use double enzymes to digest enzymes with a common buffer.
2. Preparation of target fragments.
PCR is a common molecular biology technique used to amplify and replicate specific DN** segments, which can greatly amplify a small amount of DNA to obtain a large number of target gene fragments.
3. Enzyme digestion ligation.
Once the fragment of interest is obtained, two tools must be used to ligate to the plasmid vector – an enzyme digestion tool (restriction enzyme) and a ligation tool (DNA ligase), where the endonuclease must produce the same interface to the target fragment and the plasmid vector, otherwise the DNA ligase will not be able to attach them.
Fourth, transformation identification.
Add the ligation product to competent cells (common competent cells: TOP10, DH5, BL21): place on ice for 30min, perform 42 heat shock conversion or electroconversion, then add non-resistant LB medium, incubate on a shaker at 200 rpm and 37 for 45-60 min, and then coat on a resistant or other screening plate, 37 culture overnight, pick 3-5 single clonal colonies, PCR or extract plasmids after enzyme digestion identification, After identification, the plasmid or bacterial solution is sent for sequencing verification.
That's itThe process of plasmid construction!I believe that everyone has also understood, and experimenters can design various types of plasmid vectors from different experimental purposes.