First, the primers are closely complementary to the sequence of the template, and the secondary primers cannot initiate DNA polymerization (ie, mismatch) at the non-target site of the template, and avoid formation of a stable dimer or hairpin structure between the primer and the primer. Primer design should pay attention to the following points:
1. The length of the primer is generally 15-30 bp, and the commonly used is 18-27 bp.
2. The primer sequence should have no similarity in the template, especially the sequence with higher similarity at the 3' end, otherwise it will easily lead to mismatch. The appearance of more than 3 consecutive bases at the 3' end of the primer, such as GGG or CCC, also increases the probability of error initiation.
3. The last base at the 3' end of the primer has a large effect on the DNA synthesis efficiency of the Taq enzyme. Different terminal bases result in different amplification efficiencies in the mismatched position, and the mismatch efficiency of the last base is A is significantly higher than the other three bases, so the use of base A at the 3' end of the primer should be avoided.
4. The GC content of the primer sequence is generally 40-60%, and too high or too low is not conducive to the initiation of the reaction. The GC content of the upstream and downstream primers cannot differ too much.
5. Another important parameter of the primer is the melting temperature (Tm). This is the temperature at which 50% of the primers and complementary sequences behave as double-stranded DNA molecules. Reasonable annealing temperatures range from 55 Â° C to 70 Â° C. For best results, the two primers should have an approximate Tm value.
6. Primer dimer or hairpin structure may also cause PCR reaction failure. Should be avoided as much as possible.
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