![]() ![]() Annealing temperature rules for primer sets can vary between different DNA polymerases. Use the annealing temperature recommended for a specific DNA polymerase in its optimal buffer.Adjust the annealing temperature when a PCR additive or co-solvent is used.The optimal annealing temperature is usually 3–5☌ below the lowest primer T m. Optimize the annealing temperature stepwise in 1–2☌ increments, using a gradient cycler when available.On the other hand, long denaturation times and high temperatures may reduce enzyme activity. Short denaturing times and low temperatures may not separate double-stranded DNA templates well. Optimize the DNA denaturation time and temperature.Mix the reagent stocks and prepared reactions thoroughly to eliminate density gradients that may have formed during storage and setup.Optimize the ratio of the modified nucleotide to dNTP to increase PCR efficiency. ![]()
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