The reactions were carried out as described previously [29 (link), 30 ]. In brief, a 25 μl reaction mixture contained 1.6 mM of each FIP and BIP primer, 0.8 mM of LB primer, 0.2 mM of each F3 and B3 primer, 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 8 mM MgSO4, 10 mM (NH4)2SO4, 0.1% Triton X–100, 0.8 M betaine (Sigma-Aldrich, St. Louis, MO), 1.4 mM dNTP mixture (New England Biolabs, Ipswich, MA), 8 U Bst DNA polymerase (New England Biolabs, Ipswich, MA), and 5 μl of DNA template. The reaction mixture was incubated at 60 °C for 60 min. The LAMP amplified DNA products were examined by electrophoresis on 2% agarose gel stained with a 1:10,000 dilution of GelRed (Phenix Research Products, Asheville, NC) and visualized using UV light. Since the detecting reagent SYBR green was present in the lyophilized reagents, the results were visualized directly from the reaction vial with a UV lamp. At each storage time point, the assays were performed at 100, 50, 25, and no copies of pET24-IS1111a plasmid in duplicate.
Dntp mixture
Manufactured by New England Biolabs
The DNTP mixture is a solution containing the four deoxyribonucleotide triphosphates (dATP, dCTP, dGTP, and dTTP) required for DNA synthesis. The mixture is a fundamental component in various molecular biology techniques, such as DNA amplification, sequencing, and labeling.
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Lab products found in correlation
2 protocols using dntp mixture
1
LAMP Assay for Plasmid Detection
2
LAMP Assay for Rapid Detection
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LAMP reactions were carried out as described previously [11 (link)]. Briefly, a 25 μL reaction mixture contained 1.6 mM of each FIP primer and BIP primer, 0.8 mM of each LF primer and LB primer, 0.2 mM of each F3 primer and B3 primer, 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 8 mM MgSO4, 10 mM (NH4)2SO4, 0.1% Triton X-100, 0.8 M betaine (Sigma-Aldrich, St. Louis, MO), 1.4 mM dNTP mixture (New England Biolabs, Beverly, MA), 8 U Bst DNA polymerase (New England Biolabs, Beverly, MA), and 5 μL of DNA template. The optimal reaction temperature of 63°C was experimentally determined by varying the temperature from 58 to 63°C. The reaction mixture was incubated for 60 min. Each reaction was terminated by adding 5 μL of 10X BlueJuice (Invitrogen, Carlsbad, CA) for gel detection. The reaction products were examined by electrophoresis on a 2% agarose gel stained with a 1 : 10,000 dilution of GelRed (Phenix Research Products, Asheville, NC). Other detection methods involved inclusion of dyes before amplification, such as addition of HNB [18 (link)] into the reaction to enable direct visual detection or inclusion of SYBR green to detect the reaction products by a UV light.
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