A dual-purpose buffer for fluorescent dye and electrophoresis PCR

Abstract

The invention discloses a fluorescent dye and electrophoretic PCR dual-purpose buffer, which comprises the following components: sodium cresyl red (1.5-2.5)*10^-5g / L, PCR Buffer 71-75%, dNTP 5.5-6.5 %, glycerin 4-5%, SYBR Green Ⅰ 0.008-0.015% and sterilized deionized water 15-20%. The dual-purpose dye of the present invention has the versatility of different detection platforms, and can be applied to Taq enzymes of different brands. At least it has been confirmed that it can be used for Invitrogen, Promega, Bioer Taq and pfu Taq, and can be used for Syber green I real-time PCR amplification at the same time. Increased or general PCR amplification, and after the fluorescent PCR amplification reaction, agarose electrophoresis can be performed directly to perform product recovery or interpretation, without adding additional dyes for electrophoresis, and electrophoresis detection can still be performed after fluorescent PCR detection without additional addition stain. The buffer formula can simplify the two processes of diluting the dye and adding the nucleic acid dye during the electrophoresis operation. In particular, carcinogens such as traditional nucleic acid dye EtBr are omitted, which greatly increases the safety of the test.

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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