NEB Q5 PCR

(Q5 is a high fidelity polymerase supplied by New England Biolabs)

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1. Calculate Tm and annealing temperatures of primer pairs using the <a href="https://www.neb.com/tools-and-resources/interactive-tools/tm-calculator">NEB Tm Calculator</a>.
   NOTE: If your primers are not entirely complementary to the template, remember to calculate annealing temperatures for the first annealing reaction (primer to template) not the whole primer length.
   
   
2. Dilute each lyophilized primer (from IDT) with nuclease-free water to get solutions of 100 μM.
   e.g. if 80.8 nanomoles of primer, add 0.808 ml of nuclease-free water.
   Make up dilution in tube primer delivered in. Pipette up and down then spin gently in bench centrifuge to thoroughly mix. NOTE: Do not contaminate water filter nozzle when collecting nuclease-free water.
   
   
3. Add 45 μl of nuclease free water to a 1.5 ml Eppendorf. Add 5 μl of a primer to give a 1 in 10 dilution (10 μM). Repeat in a fresh Eppendorf for each primer. Mix thoroughly.
   NOTE: Change tip before replacing pipette into nuclease-free water to prevent contamination of all following samples.
   
   
4. For a 25 μl PCR reaction add the following to PCR tubes on ice and pipette up and down to mix:
   File:Q5 PCR table.jpg
   
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