When designing primers, how should the protective base in front of the digestion site be added and w

When we construct the vector, we generally add 2 to 3 bases in front of the enzyme cleavage site, and the base is generally G or C.

It seems to be according to a specific sequence...

1. Add additional base sequences, that is, protect bases, to improve the activity of enzymatic digestion in the future. When adding a protective base, two factors need to be considered: one is the number of bases, and the other is the type of base. Different endonucleases require minimum protection of base numbers other than the recognition site.

2. Two adjacent digestion sites are not used at the same time. When selecting a vector cleavage site in a molecular cloning experiment, two adjacent cleavage sites often cannot be used at the same time, because one site cleavage leaves too few bases to affect the cleavage of the adjacent cleavage site.

DNA synthesis, the extension direction of the new strand is 5 3

Therefore, it is necessary to add the enzyme digestion site at the 5th end, remember to add some protective bases, because in addition to the specific recognition site, the endonuclease also needs several more non-specific bases to provide a platform so that it can bind up, otherwise it will fall off.

The specific number of protected bases is generally 5-6.

The structure of the primer should be (5 3): protection base + enzyme cleavage site + original primer sequence.

When designing primers for overexpression vectors, the digestion sites need to be counter-complementary.

Explanation: Restriction sites are specific DNA sequences recognized by restriction enzymes, which are important molecular biology tools. In the primer design of the overexpression vector, it is necessary to insert the enzyme cleavage site so that the restriction digestion can be performed during the cloning process, so that the target gene can be correctly inserted into the worm-carrying body.

The enzyme cleavage site needs to be counter-muffled and concealed complementary because the DNA sequence recognized by the restriction enzyme is double-stranded and symmetrical, so the pairing of reverse complementation needs to be considered when designing primers to ensure the correctness of the enzyme cleavage site.

Practical solutions and countermeasures: When designing primers for overexpression vectors, you can use some professional soft withered mustache tools, such as primer3, snapgene, etc., which can automatically search for and design primers that meet the requirements, and automatically calculate the reverse complementary pairing of enzyme cleavage sites. In addition, you can also refer to relevant literature and databases, such as nebcutter, rebase, etc., to determine the suitable digestion site for your experiment.

Extended note: In laboratory research, the design of primers for overexpression vectors is a very important step, and its quality directly affects the final result. Therefore, when designing primers, multiple factors need to be considered, such as primer length, TM value, GC content, complementarity, etc., to ensure the specificity and stability of primers.

At the same time, care should also be taken to avoid non-specific binding of primers to vectors or other non-target genes, which can affect the experimental results.

When designing primers for overexpression vectors, the digestion sites need to be reversed complementary. This is because the overexpression vector primer design requires the use of restriction enzymes to insert the gene of interest into the vector. Restriction endoceases can recognize and cleave specific DNA sequences, which are usually symmetrical and present on both complementary DNA strands.

Therefore, when designing primers for overexpression vectors, it is necessary to consider the digestion sites recognized by restriction enzymes in the design of primers. To ensure that primers can be properly attached to the vector, reverse complementary cleavage sites need to be designed. In this way, the restriction bent curvature enzyme can properly cleave the DNA sequence and insert the gene of interest into the vector as the primer binds to the vector.