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. Prolong the extension time according to amplicon lengths.Reduce the annealing and extension temperatures to help primer binding and enzyme thermostability.Choose DNA polymerases with high processivity, which can amplify long targets in a shorter time.Use DNA polymerases specially designed for long PCR. Check amplification length capability of the selected DNA polymerases.Increase denaturation time and/or temperature to efficiently separate double-stranded DNA templates.Use a PCR additive or co-solvent to help denature GC-rich DNA and sequences with secondary structures.Choose DNA polymerases with high processivity, which display high affinity for DNA templates and are more suitable to amplify difficult targets.If appropriate, increase the number of PCR cycles.Ĭomplex targets (e.g., GC-rich or secondary structures).Choose DNA polymerases with high sensitivity for amplification.Examine the quantity of input DNA and increase the amount if necessary.

Choose DNA polymerases with high processivity, which display high tolerance to common PCR inhibitors carried over from soil, blood, plant tissues, etc.Re-purify, or precipitate and wash DNA with 70% ethanol, to remove residual salts or ions (e.g., K +, Na +, etc.) that may inhibit DNA polymerases.Ensure that no residual PCR inhibitors such as phenol, EDTA, and proteinase K are present if following chemical or enzymatic DNA purification protocols.Consult the user manual and troubleshooting guides to mitigate poor DNA quality. Follow manufacturer recommendations stringently when using purification kits to isolate template DNA.Store DNA in molecular-grade water or TE buffer (pH 8.0) to prevent degradation by nucleases.Evaluate template DNA integrity by gel electrophoresis, if necessary. Minimize shearing and nicking of DNA during isolation.

Pfu ultra touchdown pcr