Loopamp dna amplification reagent kit

The reactions were performed using a Loopamp DNA Amplification Reagent Kit (Eiken Chemical). Detection of the LAMP amplicons was performed by real-time measurements of turbidity using a Loopamp real-time turbidimeter LA-200 (Eiken Chemical) and visual observations of the colour changes with the naked eye under natural light. The final reaction volume was 25 μL, comprising 40 pmol of the FIP primer and 40 pmol of the BIP primer, 20 pmol of each loop primer, 5 pmol of F3 and B3 primers, 1 μL of the Bst DNA polymerase, reaction buffer (20 mM Tris–HCl, 10 mM KCl, 8 mM MgSO₄, 10 mM (NH₄)₂SO₄, 0.1% Tween 20, 0.8 M betaine, and 1.4 mM each dNTP), and 1 μL of plasmid DNA or 8 μL of genomic DNA samples. Reactions were carried out at 62, 63, 64, and 65 °C in duplicate to find the optimum temperature for the LAMP amplification.

The reactions were performed using a Loopamp DNA Amplification Reagent Kit (Eiken Chemical), detection was performed by real-time measurement of turbidity using a Loopamp EXIA (Eiken Chemical), and visual observation with the naked eye. DNA samples (1 μL) were subjected to amplification in reaction mixtures, each of which contained 40 pmol of the FIP and BIP primers, 20 pmol of the each loop primer, 5 pmol of F3 and B3 primers, 1 μL of provided Bst DNA polymerase, and reaction buffer (20 mM of Tris-HCl, 10 mM of KCl, 8 mM of MgSO₄, 10 mM of (NH₄)₂SO₄, 0.1% of Tween 20, 0.8 M of betaine, and 1.4 mM of each dNTP), with or without 1 μL of fluorescent detection reagent (FDR) (Eiken Chemical). Distilled water was added to bring the final volume of 25 μL. Successful targeted gene amplifications without FDR were detected by real-time measurement of turbidity with a Loopamp EXIA, and those with FDR were detected by the naked eye from color changes under natural light. Reactions were carried out at 61°C, 62°C, 63°C, and 64°C in triplicate to find the optimum temperature for LAMP amplification.

The reactions were performed using a Loopamp DNA Amplification Reagent Kit (Eiken Chemical). Detection of the LAMP amplicons was performed by real-time measurements of turbidity using Loopamp EXIA (Eiken Chemical) and visual observation of color changes with the naked eye under natural light. The final reaction volume was 25 μL, comprising 80 pmol of the FIP primer and 40 pmol of the BIP primer, 20 pmol of each loop primer, 5 pmol of F3 and B3 primers, 1 μL of the provided Bst DNA polymerase, reaction buffer (20 mM Tris-HCl, 10 mM KCl, 8 mM MgSO₄, 10 mM (NH₄)₂SO₄, 0.1% Tween 20, 0.8 M betaine, and 1.4 mM each dNTP), and 1 μL of plasmid DNA or 1–8 μL of genomic DNA samples. Reactions were carried out at 62 °C, 63 °C, 64 °C, and 65 °C in duplicate to find the optimum temperature for LAMP amplification.