2. Important Notes
• The recommended starting amount is 5% blood
added directly to the reaction without further
modification.
• Use 98°C for denaturation.
• The annealing rules are different from many common
DNA polymerases (such as Taq DNA polymerases).
Read Section 5.2 carefully.
• For extension, use 15 s for amplicons ≤1 kb or
30 s/kb for amplicons >1 kb.
• After PCR, spin the reactions at 1 000 × g
(about 4 000 rpm) for 1−3 minutes to pellet debris from
blood.
• For blood concentrations greater than 10%,
optimization of MgCl2 concentration may be required
(see 4.3).
• Phusion DNA Polymerases produce blunt end PCR
products.
3. Guidelines