E z n a blood dna kit

DNA was analyzed for the presence for the presence of one of the common G6PD mutations G → A at nt 202 using Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. DNA was extracted from a prepared buffy coat using E.Z.N.A blood DNA kits (Omega Bio-Tek Inc. Doraville. GA 30362. USA). PCR amplification was done using primers F – 5’-CCA CCA CTG CCC CTG TGA CCT-3’ and R- 5’-GGC CCT GAC ACCACC CAC CTT-3’. Details of the PCR-RFLP process are described elsewhere [9 (link)].
Thick blood smears were done to assess for malaria parasites using equal volumes of venous blood and stained with 2% Giemsa. All the blood smears were then double read independently by trained laboratory technicians. Where results between the two expert microscopists were discordant, a third microscopist re-read the blood slide and the majority decision was accepted as the final result. Parasite densities were determined from thick blood smears by examining the smears under × 100 objective. The average number of parasites/oil immersion field was then determined and the parasite density in number/μl was calculated by multiplying the average number of parasites per field by a microscope factor of 1/0.002. Samples collected at baseline were used to determine prevalence and G6PD status of the participants.

Genomic DNA was extracted from blood leukocytes using E.Z.N.A Blood DNA kit as outlined by the manufacturer’s protocol (Omega Bio-tek, USA). Approximately 0.1 µg of genomic DNA was used for the genotyping assays.