<ul><li>A high quality, purified DNA template is preferred as it greatly improves PCR success. Recommended amounts of such a template are shown below for a 50uL reaction:</ul></li>
<ul><li>Oligonucleotide primers should generally be 20-40 nucleotides long while having a GC content of 40-60%</li>
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<li>Best results are seen when using each primer at a final concentration of 0.5uM in the reaction</li></ul>
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Mg2+ and additives
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<ul><li>The Q5 High-Fidelity Master Mix contains 2mM Mg++ when used at a 1X concentration, which is optimal for most PCR products</li></ul>
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Deoxynucleotides
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<ul><li>Final concentration of dNTPs is 200uM of each deoxynucleotide in the 1X final concentration</li>
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<li>Q5 High-Fidelity DNA Polymerase cannot incorporate dUTP and isn’t recommended for use with uracil-containing primers or templates</li></ul>
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Q5 High-Fidelity DNA Polymerase concentration
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<ul><li>Concentration in the Master Mix has been optimized for best results under a wide conditions range</li></ul>
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Denaturation
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<ul><li>Initial denaturation of 30 seconds occurs at 98oC, which is enough for most amplicons from pure DNA templates.</li>
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<li>Though longer denaturation times going up to 3 minutes can be used for templates that require it</li></ul>
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Annealing
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<ul><li>Optimal annealing temperatures for this Master Mix tend to be higher than for other PCR polymerases</li>
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<li>Typically 10-30 second annealing steps should be used at 3oC above the Tm of the lower Tm primer</li>
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<li>Temperature gradients can also be used to optimize the annealing temperature for each primer pair<ul>
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<li>For higher Tm primer pairs, two-step cycling without a separate annealing step can be used</li></ul></li></ul>
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Extension
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<ul><li>Recommended extension temperature is 72oC
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o With the recommended time being between 20-30 seconds per kb for complex, genomic samples.
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• The time can be reduced to 10 seconds per kb for simpler templates (plasmid, E.coli, etc.) or complex templates smaller than 1kb
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• The extension time can be increased to 40 seconds per kb for cDNA or other long, complex templates if needed
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• A final extension of 2 minutes at 72oC is recommended</li></ul>
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Cycle Number
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<ul><li>25-35 cycles yield sufficient products generally
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• For genomic amplicons, 30-35 cycles are advised</li></ul>
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2-step PCR
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<ul><li>Used when primers have annealing temperatures exceeding or are equal to 72oC (≥ 72°C).
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• This 2-step thermocycling protocol combines annealing and extension into one step</li></ul>
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Amplification of long products
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<ul><li>When amplifying products > 6kb, you can increase the extension time to 40-50 seconds per kb.</li></ul>
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PCR Product
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<ul><li>Products generated using this Master Mix have blunt ends
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• If clonding is the next step then blunt-end cloning isn’t recommended
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• If T/A-cloning is to be done, the DNA should be purified prior to A-addition, since the Q5 High-Fidelity DNA Polymerase will degrade any overhangs generated</li></ul>
Experiments & Protocols
