Letter of apology

Letter of apology
PCR reaction primer design the basic requirements of the basic requirements of the primers are: 1. The length of the primer is too short will affect PCR specificity required 16-30bp, because 4 ^ 16 = 4.29x10 ^ 9, is greater than the mammalian genome 3x10 ^ 9bp, to ensure that the specific binding; primer extension temperature is too long so that Taq DNA polymerase than the optimal temperature (74 degrees), will also affect the specific products. 2. G + C content is typically 40% -60%. 3. The four bases should be randomly distributed, do not have more than three consecutive purine or pyrimidine same presence. Especially the primer 3 'end, there should cofares be three consecutive G or c, otherwise it would be a nucleic acid primer or c G-rich region complementary error, affecting cofares the specificity of PCR. 4. primers complementary sequence itself should not cause their fold, at least primers complementary bases can not be greater than their continuous cofares 3bp. 5. Between the two primers should not be complementary, especially their 3 'ends should not be complementary. Should not be more than between a pair of primers 4 consecutive bases are complementary, in order to avoid primer-dimer. cofares 6. homology with non-specific primer between the target do not exceed 70% or 8 consecutive complementary bases homologous otherwise cause non-specific amplification. 7. The primer 3 'end is extended trigger point. Therefore should not be a mismatch. ATCG caused due to a mismatch there are certain rules to the primer 3 'end A greatest impact, therefore, try to avoid the primer 3' end of the first base is an A. The primer 3 'end of the codon encoding if no third base, in order to avoid because the first three codon degeneracy affect amplification specificity. 8. primers 5 'end can be modified, including the add restriction sites, biotin, a fluorescent substance, digoxin and other markers, introducing a mutation site, a promoter sequence is introduced, the introduction of a protein binding DNA sequences and the like, most primers were designed good guide to computer software. Reaction system, primer concentrations generally require between O.1-0.5μmol. Concentration is too high, easy to generate primer dimers or nonspecific products. Tm value of primer annealing temperature, is calculated as follows: Tm = 4 (G + C) +2 (A + T). Primer Tm value is preferably in the range of 55-80 , close to 72 preferred. cofares
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