With the usual design of degenerate m13 primers differ by CodeHop designed m13 degenerate m13 prime

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Because the relationship between the codons, the amino acid sequences of different nucleic acid sequences may express the same, so the amino acid sequence is the real conservative. Firstly, NCBI's Entrez retrieval system, m13 you can find a related sequences. Subsequently using this sequence using BLASTp (check by protein protein), find with similar amino acid sequences throughout Nr database.
Once obtained conserved regions, you can use specialized software to design primers, such as the use of degenerate primers were designed pp5 matter. The participation of multiple sequence alignment according to any one of the sequences in the import pp5, and then translated into a nucleotide sequence, there is codon degeneracy, the result is only a difference of a plurality of n nucleotide m13 sequence m13 of each group The group have available a degeneracy of nucleotide chains (wherein R = A / G, Y = C / T, M = A / C, K = G / T, S = C / G, W = A / T, H = A / C / T, B = C / G / T, V = A / C / G, D = A / G / T, N = A / C / G / T), which has a simple and a nucleotide m13 chain must contain the corresponding part of the amino acid conserved region found in the previous step, modify parameters pp5, and make the appropriate distance in two conserved regions to find nt primer pairs, in short, to ensure that the downstream primer fall within the degenerate chain of conserved regions, the result will be the number of pairs, the higher the score the better.
Y represents C or T
With the usual design of degenerate m13 primers differ by CodeHop designed m13 degenerate m13 primers. Primer consists of two parts: a primer for the 3 end of the 4-5 conservative amino acid according to the design of degenerate core region (3 core region), the length of only 11-15 bases; primers 5 'end non-degeneracy of the splint structure (5 consensus clamp region). 5 end plywood structure is based on the consensus sequences designed codon bias, its greatest extent predicted coding sequences conserved amino acids, and its length depending m13 on the desired annealing temperature. The advantage of this design is that not only reduces the degenerate primers and degrees, but also improves the primer annealing temperature, protect the specificity of the PCR products. Standard degenerate PCR in the late polymerization reaction, with the increase of products, non-specific binding phenomenon primer increased; and late CodeHop PCR, this phenomenon is greatly reduced. Experimental results m13 show that by this method designed degenerate primers to reduce non-specific amplification, is a fast and convenient design degenerate primers.
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