Fluorescent dye and electrophoresis PCR (Polymerase Chain Reaction) dual-purpose buffer solution

Abstract

The invention discloses a fluorescent dye and electrophoresis PCR (Polymerase Chain Reaction) dual-purpose buffer solution which comprises the following components: (1.5-2.5)*10<-5>g / L of sodium cresol red, 71-75% of PCR Buffer, 5.5-6.5% of dNTP, 4-5% of glycerin, 0.008-0.015% of SYBR Green I and 15-20% of sterilized deionized water. The dual-purpose dye has universality to different detection platforms, can be applicable to Taq polymerase of different brands, is at least confirmed to be applied to Invitrogen, Promega, Bioer Taq and pfuTaq, and can be simultaneously applied to Syber green I real-time PCR amplification or general PCR amplification. Moreover, agarose electrophoresis can be directly performed to execute product recovery or interpretation after the fluorescent PCR amplification, electrophoresis can be performed without adding an extra dye, electrophoretic determination can also be performed after fluorescent PCR detection, and no extra dyeing agent is need to be added. According to a formula of the buffer solution, two processes of diluting the dye and adding nucleic acid dyes during electrophoretic procedures can be simplified, particularly the traditional nucleic acid dye EtBr and other cancerogens are omitted, and the test safety is greatly improved.

Description

technical field [0001] The technical field of molecular testing of the present invention, in particular relates to a dual-purpose buffer solution for fluorescent dyes and electrophoresis PCR. Background technique [0002] The addition of a buffer in the PCR reaction helps to stabilize the enzyme, and can provide the ions required for the activity of the polymerase and a suitable pH, providing the most suitable conditions for the enzyme-catalyzed reaction to the PCR reaction. Glycerol and sodium cresyl red are generally not added to the buffer used in traditional Real time PCR. It is generally believed that sodium cresyl red will absorb Syber green I to disperse fluorescence and reduce the fluorescence value, while the addition of glycerol will cause the solution density to increase and affect the fluorescence value. When there is Sybergreen I in the buffer, because the density of the PCR reaction solution is smaller than that of the buffer used for electrophoresis during el...

AI Technical Summary

Problems solved by technology

It is generally believed that sodium cresyl red will absorb Syber green I to scatter fluorescence and reduce the fluorescence value, while the addition of glycerol will cause the solution density to increase and affect the fluorescence value

When there is Sybergreen I in the buffer, because the density of the PCR reaction solution is smaller than that of the buffer used for electrophoresis during electrophoresis, it will float up when the reaction solution is added, and the sample cannot be loaded. The PCR reaction solution is a colorless and transparent liquid, which makes it difficult to load the sample. Generally, the current electrophoresis method needs to dilute and mix the PCR reaction solution and the dye (including a solution that increases the density) to load the sample smoothly.

[0003] When the concentration of Syber green I used in Real time PCR is low, if the nucleic acid dye is not added to the agarose gel during electrophoresis or the counterstaining process after electrophoresis, the PCR amplification bands cannot be successfully interpreted

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