Journal: PLoS ONE
Article Title: An Efficient Strategy for Broad-Range Detection of Low Abundance Bacteria without DNA Decontamination of PCR Reagents
Figure Lengend Snippet: HotStart and low-DNA Taq DNA polymerases are not sufficiently pure for sensitive and specific broad-range amplification of bacterial DNA. The genomic DNA (100 fg) of S. aureus was amplified by HotStart or low-DNA Taq DNA polymerases (Taq #1: Hot Start Taq DNA polymerase, Protech Inc.; Taq #2: Fast Hot Start Taq DNA polymerase, KAPA Biosystems; Taq #3: Taq DNA polymerase, TakaRa Inc.; Taq #4: ULTRATOOLS Taq DNA polymerase, Biotools Inc.) using the primer set p201 and p1370 (lanes 1, 3, 5, and 7). Significant amount of PCR product was present in the no template control reactions (lanes 2, 4, 6, and 8).
Article Snippet: Pretreatment of DNase I for decontamination and broad-range PCR amplification The PCR reagents containing 2 µl of 10× BSA, 2 µl of dNTP (2 mM), 2 µl of 10× LCGreen I plus, 1 µl of 10× PCR buffer, 0.5 µl of HotStart Taq DNA polymerase (5 U/µl), were incubated with DNase I (1 U or 2.5 U) at 37°C for 30 min. After heat inactivation of DNase I at 85°C for 15 min, the reaction mixture was brought up to 20 µl by adding 1 µl of 10× PCR buffer, 2 µl of forward primer p201 (5 µM), 2 µl of reverse primer p1370 (5 µM), and 5 µl of template DNA.
Techniques: Amplification, Polymerase Chain Reaction