RT-LAMP Fluorenscent Master Mix

RT-LAMP Fluorenscent Master Mix is all-in-one RT-LAMP kit with fluorescent detection: Mix buffer, enzymes, dye, add primers and template. High efficiency, sensitivity for DNA/RNA isothermal amplification.

Cat No: HCB5205A

Package: 96RXN/960RXN/9600RXN

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Product Description

Product detail

Product Tags

This product contains reaction buffer, RT-Enzymes Mix (Bst DNA polymerase and heat-resistant reverse transcriptase), lyophilized Protectants and fluorescent dye components. The reaction buffer contains Mg2+, dNTP and other necessary components for amplification. To use, just mix the Buffer, reaction enzyme, fluorescent dye, primer and add the template, adding lyophilized protectant can use straightly. It was connected to a lyophilizer and lyophilized, and only the primers and templates were added when used. This reagent has high amplification efficiency and sensitivity.

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Component

Component	HCB5205A-01	HCB5205A-02	HCB5205A-03
Loop-mediated Amplification Buffer	1.2 mL	4 mL×3	12 mL×10
RT-Enzymes Mix	245 μL	2.45 mL	2.45 mL×10
Lyophilized protectant	0.96 mL×2	9.60 mL×2	9.60 mL×20
25× Dye	192 μL	0.96 mL×2	0.96 mL×20

Applications

For DNA or RNA isothermal amplification.

Storage Conditions

Transported with Dry ice, stored at -25~ -15℃. Avoid frequent freeze-thaw, the product is valid for 12 months.

Protocol

1.Thaw the reaction buffer to be used at room temperature. Vortex briefly or invert tubes several times to mix thoroughly, then centrifuge to collect the liquid to the bottom of the tube.

2.Preparation of reaction system. This reagent can be prepared in two reaction systems, liquid reaction mix and lyophilized system mix.

1) Prepare liquid reaction mix

Component	Volume
Loop-mediated Amplification Buffer	12.5 μL
25× Dye	2 μL
RT-Enzymes Mix	2.55 μL
10 × Primer Mix ^a	5 μL
Templates DNA/ RNA^b	× μL
Nuclease-free Water	Up to 50 μL

2) Lyophilization system mix

①　Prepare lyophilized mix

Component	Volume
Loop-mediated Amplification Buffer	12.5 μL
Lyophilized protectant	20 μL
25× Dye	2 μL
RT-Enzymes Mix	2.55 μL
Nuclease-free Water	Up to 50 μL

②　Lyophilization: The prepared Mix was lyophilized in a 50μL system

③　Prepare reaction mix

Component	Volume
Lyophilized mix	1 piece
10 × Primer Mix ^a	5 μL
Templates DNA/ RNA^b	× μL
Nuclease-free Water	Up to 50 μL

Notes:

1) a. 10×Primer Mix : 16 μM FIP/BIP, 2 μM F3/B3, 4 μM Loop F/B;

2) b. DEPC (water soluble) is recommended for nucleic acid templ.

3) Amplification Reaction: Set up the following program on a fluorescent PCR instrument (e.g. ABI7500, etc.):

Temperature	Time	Circles
65 ℃	60 sec *collect FAM fluorescence	30

Notes

1.Salt may appear in the bottom of the buffer tube, vortex briefly or invert tubes several times to mix thoroughly at room temperature.

2.The reaction temperature can be optimized between 62 ℃ and 68 ℃ according to the condition of primers.

3.The experiment should be standardized, including the preparation of the reaction system, lyophilization, and sample processing and sample adding process;

4.To avoid contamination, it is recommended to prepare the reaction system in an ultra-clean bench, in other Add templates to the fume hood of the room to avoid false positive interference.