Phi29 polymerase

The sWGA reactions were performed using one set of primers for P. ovale curtisi and one set of primers for P. ovale wallikeri. Each reaction was performed in 0.2 mL PCR tubes containing at least 20 ng of template genomic DNA, 0.1 mg/mL bovine serum albumin (BSA) (New England Biolabs), 1 mM dNTPs (New England Biolabs), 2.5 μM each primer, 1× Phi29 reaction buffer (New England Biolabs), 30 units of Phi29 polymerase (New England Biolabs), and molecular biology-grade water to reach a final reaction volume of 50 μL. The reaction was carried out on a thermocycler (Mastercycler Gradient, Eppendorf) with the following step-down program: 5 min at 35°C, 10 min at 34°C, 15 min at 33°C, 20 min at 32°C, 25 min at 31°C, 16 h at 30°C, then heating for 15 min at 65°C to inactivate the Phi29 polymerase before cooling to 4°C. Amplified products were quantified using the Qubit dsDNA high-sensitivity kit (Thermo Fisher Scientific). Amplified products were cleaned using Agencourt Ampure XP beads (Beckman Coulter) as follows: 1.8 volumes of beads were added to 1 volume of amplified products, briefly mixed, and then incubated for 5 min at room temperature. A magnetic rack was used to capture the DNA binding beads. The beads were then washed twice using 200 μL of 80% ethanol, and DNA was eluted with 60 μL of EB buffer.

The sWGA reactions were performed using one set of primers for P. ovale curtisi and one set of primers for P. ovale wallikeri. Each reaction was performed in 0.2 mL PCR tubes containing at least 20 ng of template genomic DNA, 0.1 mg/mL bovine serum albumin (BSA) (New England Biolabs), 1 mM dNTPs (New England Biolabs), 2.5 μM each primer, 1× Phi29 reaction buffer (New England Biolabs), 30 units of Phi29 polymerase (New England Biolabs), and molecular biology-grade water to reach a final reaction volume of 50 μL. The reaction was carried out on a thermocycler (Mastercycler Gradient, Eppendorf) with the following step-down program: 5 min at 35°C, 10 min at 34°C, 15 min at 33°C, 20 min at 32°C, 25 min at 31°C, 16 h at 30°C, then heating for 15 min at 65°C to inactivate the Phi29 polymerase before cooling to 4°C. Amplified products were quantified using the Qubit dsDNA high-sensitivity kit (Thermo Fisher Scientific). Amplified products were cleaned using Agencourt Ampure XP beads (Beckman Coulter) as follows: 1.8 volumes of beads were added to 1 volume of amplified products, briefly mixed, and then incubated for 5 min at room temperature. A magnetic rack was used to capture the DNA binding beads. The beads were then washed twice using 200 μL of 80% ethanol, and DNA was eluted with 60 μL of EB buffer.