Bacterial DNA was extracted using a QIAamp DNA Mini Kit
(QIAGEN Inc., Valencia, CA), following the manufacturer’s instructions
(Appendix D: Protocols for Bacteria). Briefly, 180 mL of an
enzyme solution (20 mg/mL lysozyme; 20 mM TriseHCl, pH 8.0;
2 mM EDTA; 1.2% Triton) was put into the tube of the blood sample
and then vortexed. The bloodstain sample was moistened with
180 mL of the enzyme solution in the tube and mixed well by
agitation with a pipet tip. After incubation for 30 min at 37 C, 25 mL
proteinase K and 200 mL Buffer AL were added to each tube and the
tubes were mixed by vortexing. After incubation for 30 min at
56 C, 200 mL ethanol (99.5%) was added and the tubes were mixed
thoroughly by vortexing. The following step was performed according
to the manufacturer’s instructions (Protocol: DNA Purification
from Tissues) and the purified bacterial DNA was finally
eluted in 100 mL Buffer AE. The DNA solution was stored at 30 C
until use. As an extraction negative control, 180 mL of the enzyme
solution alone was used.
Bacterial DNA was extracted using a QIAamp DNA Mini Kit(QIAGEN Inc., Valencia, CA), following the manufacturer’s instructions(Appendix D: Protocols for Bacteria). Briefly, 180 mL of anenzyme solution (20 mg/mL lysozyme; 20 mM TriseHCl, pH 8.0;2 mM EDTA; 1.2% Triton) was put into the tube of the blood sampleand then vortexed. The bloodstain sample was moistened with180 mL of the enzyme solution in the tube and mixed well byagitation with a pipet tip. After incubation for 30 min at 37 C, 25 mLproteinase K and 200 mL Buffer AL were added to each tube and thetubes were mixed by vortexing. After incubation for 30 min at56 C, 200 mL ethanol (99.5%) was added and the tubes were mixedthoroughly by vortexing. The following step was performed accordingto the manufacturer’s instructions (Protocol: DNA Purificationfrom Tissues) and the purified bacterial DNA was finallyeluted in 100 mL Buffer AE. The DNA solution was stored at 30 Cuntil use. As an extraction negative control, 180 mL of the enzymesolution alone was used.
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