Precautions for PCR tube

primer

Primers are the key to the success or failure of PCR reaction. To ensure the accuracy and efficiency of amplification, the design of primers should follow the following principles:

The same sequence of genes in different regions can be obtained by searching the same sequence of genes in different species through NCBI software

Primer length: 15-28bp, longer than 38bp, will increase the annealing temperature, which is not conducive to the amplification of common Taq enzyme

The GC content of primers is 40% - 60%, and the TM value is best close to 72 ℃

Due to the degeneracy of codon, the 3 'end of primer should avoid the third position of codon (preferably T)

The base distribution is scattered, with no more than 3 consecutive g or C at the 3 'end

The primer itself should not form a hairpin structure. After the primer design is completed, it should be verified by blast