Ensure that the selected DNA polymerases are able to incorporate the modified nucleotides.Consider using an additive or co-solvent specifically formulated for a given DNA polymerase (e.g., GC Enhancer supplied with Invitrogen Platinum DNA polymerases).ĭUTP or modified nucleotides in reaction mix.Increase the amount of DNA polymerase, or use DNA polymerases with high processivity.Adjust the annealing temperatures, as high concentrations of PCR additives or co-solvents weaken primer binding to the target.Use the lowest possible concentration when appropriate. Review the recommended concentrations of PCR additives or co-solvents.For example, Pfu DNA polymerase works better with MgSO 4 than with MgCl 2. Check the DNA polymerase’s preference for magnesium salt solutions.The presence of EDTA, other metal chelators, or atypically high concentrations of dNTPs may require a higher Mg 2+ concentration. Optimize Mg 2+ concentration for maximum PCR yields.Increase the amount of DNA polymerase if the reaction mixture contains a high concentration of an additive (e.g., DMSO, formamide) or inhibitors from the sample sources.Review recommendations on the amount of DNA polymerase to use in PCR, and optimize as necessary.Alternatively, set up PCR on ice, or add DNA polymerase last to the reaction mixture.Hot-start DNA polymerases also increase yields of the desired PCR products by eliminating nonspecific amplification. Use hot-start DNA polymerases to prevent degradation of primers by the 3’→5’ exonuclease activity of proofreading DNA polymerases.For long PCR and PCR with degenerate primers, start with a minimum concentration of 0.5 μM.Optimize primer concentrations (usually in the range of 0.1–1 μM).Reconstitute fresh primer aliquots, or obtain new primers if necessary.Aliquot primers after resuspension and store properly.Verify that the primers are complementary to the correct strands of the target DNA.Ensure that the primers are specific to the target of interest.Use online primer design tools when appropriate.
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