Editing FTA Card PCR (section)

== DNA extraction from DBSs ==

For single-stranded DNA (ssDNA) extraction, DBSs on the cards were cut into small pieces with scissors and transferred into 1.5-mL microtubes to which 500 μL dH2O had been added; the tubes were kept at room temperature for 5 minutes. The dH2O was discarded, and the procedure was repeated twice. The filter papers were soaked in 500 μL extraction buffer [phosphate-buffered saline (PBS) or 10 mM Tris-EDTA (TE)].

For lysis, red blood cell lysis buffer (100 μL) was added to the filter papers, which were then incubated with 10 μL (1 mg/mL) proteinase K at 37°C overnight. After blood cell lysis, the filter papers were pressed against the bottom of the tube several times with a clean pipette tip [20]. For increased extraction efficiency, cell lysates were incubated at 95°C for 15 minutes. Filter papers were removed after brief centrifugation (2–3 seconds), and the DNA-containing supernatant was stored at −80°C until analysis.

For double-stranded DNA (dsDNA) extraction, each filter paper sample that had been punched out was soaked in 40 μL Tris-EDTA buffer or PBS, and incubated at 37°C overnight.
[[Category:Protocols]]