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Economical and time-saving trace PCR method

A real-time fluorescence quantitative and reaction system technology, applied in the field of micro-PCR, can solve the problems of small size of DNA template, time-consuming and labor-intensive batch operation, large sample loading error, etc., to achieve the effect of saving reagents, improving precision, and saving gun tips

Pending Publication Date: 2021-12-31
WEST CHINA HOSPITAL SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Due to the small volume of DNA template added in each PCR tube, especially when it is necessary to construct a PCR reaction system with a volume of less than 20 μl, such as a 10 μl PCR reaction system, large loading errors are prone to occur
And because the master mix needs to be touched every time the DNA template is added, the pipette tip (also called the "tip") needs to be replaced every time the template is loaded. The batch operation is time-consuming and laborious, and the disposable tip is discarded directly. also cause a lot of waste

Method used

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  • Economical and time-saving trace PCR method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1, PCR reaction system loading method of the present invention

[0036] Real time PCR reaction system configuration:

[0037] Configure the Real time PCR reaction system (10μl) according to the following system: Premix Ex Taq II (2×), 5 μl; PCR forward primer (10 μM), 0.4 μl; PCR reverse primer (10 μM), 0.4 μl; template DNA (diluted 4.2 times with sterile water), 4.2 μl.

[0038] Calculate the amount of DNA template according to the number of holes, and dilute the DNA template 4.2 times with sterilized water according to the corresponding amount. And according to the number of holes made, calculate Premix Ex Taq II (2×) and primer content, according to the corresponding amount, mix the two to make a premix.

[0039] Add the master mix to the bottom of the corresponding PCR tube, 5.8 μl / tube. Then add the diluted DNA template to the middle wall of the corresponding PCR tube, 4.2 μl / tube. There is no need to change tips when adding the same DNA template....

Embodiment 2

[0041] Embodiment 2, PCR reaction system loading method of the present invention

[0042] Real time PCR reaction system configuration:

[0043] Configure the Real time PCR reaction system (20μl) according to the following system: Premix Ex Taq II (2×), 15 μl; PCR forward primer (10 μM), 1 μl; PCR reverse primer (10 μM), 1 μl; template DNA (diluted 3 times with sterile water), 3 μl.

[0044] Calculate the amount of DNA template according to the number of holes made, and dilute the DNA template 3 times with sterilized water according to the corresponding amount. And according to the number of holes made, calculate Premix Ex Taq II (2×) and primer content, according to the corresponding amount, mix the two to make a premix.

[0045] Add the master mix to the bottom of the corresponding PCR tube, 17μl / tube. Then add the diluted DNA template to the middle wall of the corresponding PCR tube, 3 μl / tube. There is no need to change tips when adding the same DNA template.

[004...

Embodiment 3

[0047] Embodiment 3, PCR reaction system loading method of the present invention

[0048] Real time PCR reaction system configuration:

[0049] Configure the Real time PCR reaction system (25μl) according to the following system: Premix Ex Taq II (2×), 15 μl; PCR forward primer (10 μM), 2 μl; PCR reverse primer (10 μM), 2 μl; template DNA (diluted 6 times with sterile water), 6 μl.

[0050] Calculate the amount of DNA template according to the number of holes made, and dilute the DNA template 6 times with sterilized water according to the corresponding amount. And according to the number of holes made, calculate Premix Ex Taq II (2×) and primer content, according to the corresponding amount, mix the two to make a premix.

[0051] Add the master mix to the bottom of the corresponding PCR tube, 19 μl / tube. Then add the diluted DNA template to the middle wall of the corresponding PCR tube, 6 μl / tube. There is no need to change tips when adding the same DNA template.

[00...

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Abstract

The invention discloses an economical and time-saving trace PCR (Polymerase Chain Reaction) method and belongs to the field of molecular biology. The method comprises the following sample adding steps: 1) adding a PCR premixed solution into the bottom of a PCR tube; 2) diluting a DNA template with water; and (3) adding all the diluted DNA template into the PCR tube, and centrifugally mixing to obtain a PCR reaction system. Preferably, in the step (3), the DNA template is added into the middle tube wall of the PCR tube. The method disclosed by the invention can be used for improving the PCR detection precision, reducing the detection error, saving the reagent and reducing the times of replacing pipettor suction tips in a smaller-volume PCR system, and is time-saving and labor-saving.

Description

technical field [0001] The invention belongs to the field of molecular biology, and in particular relates to an economical and time-saving micro-quantity PCR method. Background technique [0002] Real-time fluorescent quantitative polymerase chain reaction, called fluorescent quantitative PCR. On the basis of PCR, this detection method adds a fluorescent group, and uses the change of the fluorescent signal to monitor the process in real time, so as to achieve the purpose of relative quantification of the target gene. In the process of fluorescent quantitative PCR, it is necessary to construct a PCR reaction system (usually greater than 20 μl), including the prepared premix (enzyme mixture, primers, sterilized water, etc.) and DNA template. Adding samples to the PCR reaction system is usually to add the configured premix solution to the PCR tube first, and then add 2 μl DNA template to the premix solution respectively. [0003] Due to the small volume of DNA template added ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6851
CPCC12Q1/6851C12Q2531/113C12Q2563/107C12Q1/686C12Q1/6806B01L7/52C12Q2527/146C12Q2561/113B01L2200/0663
Inventor 王振
Owner WEST CHINA HOSPITAL SICHUAN UNIV