A. PCR Using Q5 High-Fidelity DNA Polymerase

• Note that these may differ from other polymerase protocols.
• Assemble all components on ice and transfer to preheated thermocycler. All components should be mixed prior to use.
  • Q5 High-Fidelity DNA Polymerase may be diluted in 1X Q5 Reaction Buffer just prior to use in order to reduce pipetting errors.
• Gently mix reaction. Transfer to a PCR machine and begin thermocycling.
  • Step 1: Initial denaturation, 98 degrees C, 30 seconds
  • Step 2: 25-35 cycles; 98 C, 5-10 seconds; 50-72 C, 10-30 seconds, 72 C, 20-30 seconds
  • Step 3: Final Extension, 72 C, 2 minutes
  • Step 4: Hold; 4-10 C
• General Guidelines: Use high quality, purified DNA templates to enhance success.
• Primers: Oligonucleotide primers are generally 20-40 nucleotides in length and have a GC (guanine-cytosine) content of 40-60%.
• Mg++ and additives: Mg++ concentration of 2.0 mM is usually optimal.
• Deoxynucleotides: Final concentration is usually 200 uM. (Q5 cannot incorporate dUTP and not recommended for use with uracil-containing primers/templates).
• Recommended that you use Q5 HF-DNA-P at final concentration of 20 units/mL (1 unit/50 uL reaction). Do not exceed 2 units/50 uL.