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Fast silver staining method for RCA (rolling circle amplification) product

A technique for amplified products and silver staining, which is applied in molecular biology, detection of nucleic acid amplification, and biochemistry. It can solve the problems of large amount of silver staining reagents used and inconvenient operation of changing liquids, etc., and achieves the consumption of less silver staining reagents. , easy operation and simple steps

Active Publication Date: 2018-03-23
中国医科大学
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method not only requires long-term electrophoresis operation, but also requires the gel to be placed in a large staining container for operation after electrophoresis, and the use of various silver staining reagents is relatively large, and it is necessary to prevent The damage of the gel makes it very inconvenient to change the liquid

Method used

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  • Fast silver staining method for RCA (rolling circle amplification) product
  • Fast silver staining method for RCA (rolling circle amplification) product
  • Fast silver staining method for RCA (rolling circle amplification) product

Examples

Experimental program
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Effect test

Embodiment 1

[0027] The present invention uses ordinary RCA to semi-quantitatively determine different amounts of DNA templates, and the specific steps are as follows:

[0028] (1) Design and preparation of common RCA ring template:

[0029] Design the template sequence of common RCA, prepare 10 μl ligation reaction system buffer (50 mM Tris pH 8.0, 10mM MgCl 2 , 5 mM DTT and 0.1 mM ATP), add 10 pmol linear single-stranded template DNA, 10 pmol linear single-stranded primer DNA and 175 U T4 DNA ligase, and incubate at 16 ℃ for 1 hour.

[0030] (2) RCA reaction:

[0031] Prepare 100 μl of polymerization reaction system buffer (50 mM Tris pH 7.5, 10 mM MgCl 2 , 10 mM (NH 4 ) 2 SO 4 , 4 mM DTT, 3 U phi29 DNA polymerase, 1.5~100 fmol circular template), and perform rolling circle amplification reaction.

[0032] (3) Detection of RCA products by silver staining of tube bottom glue:

[0033] ①Preparation of tube bottom glue: add 10µl of RCA amplification products with different template amounts to the bott...

Embodiment 2

[0040] The present invention uses repressor-RCA to qualitatively identify galactose and other 5 monosaccharides, and the specific steps are as follows:

[0041] (1) Design and preparation of the repressor-RCA ring template:

[0042] Design the template sequence of repressor-RCA, prepare 10 μl ligation reaction system buffer (50 mM Tris pH8.0, 10 mM MgCl 2 , 5 mM DTT and 0.1 mM ATP), add 10 pmol linear single-stranded template DNA, 10 pmol linear single-stranded primer DNA and 175 U T4 DNA ligase, and incubate at 16 ℃ for 1 hour.

[0043] (2) RCA reaction:

[0044] Prepare 100 μl of polymerization reaction system buffer (50 mM Tris pH 7.5, 10 mM MgCl 2 , 10 mM (NH 4 ) 2 SO 4 , 4 mM DTT, 3 U phi29 DNA polymerase, 0.5 pmol ligation reaction product, and galactose repressor protein 0.12 μM), each of which was added with a carbohydrate for rolling circle amplification reaction. The final concentrations of sugars are 100 μM (galactose) and 10 mM (other sugars: fructose, fucose, glucose, rib...

Embodiment 3

[0052] Example 3 The present invention detects RCA products of different reaction times

[0053] (1) Design and preparation of common RCA ring template:

[0054] Design the template sequence of common RCA, prepare 10 μl ligation reaction system buffer (50 mM Tris pH 8.0, 10mM MgCl 2 , 5 mM DTT and 0.1 mM ATP), add 10 pmol linear single-stranded template DNA, 10 pmol linear single-stranded primer DNA and 175 U T4 DNA ligase, and incubate at 16 ℃ for 1 hour.

[0055] (2) RCA reaction:

[0056] Prepare 100 μl of polymerization reaction system buffer (50 mM Tris pH 7.5, 10 mM MgCl 2 , 10 mM (NH 4 ) 2 SO 4 , 4 mM DTT, 3 U phi29 DNA polymerase, 100 fmol circular template), and perform rolling circle amplification reactions at different times.

[0057] (3) Detection of RCA products by silver staining of tube bottom glue:

[0058] ①Prepare tube bottom glue: add 10µl RCA amplification product, 3µl 40% ACR, 1µl 10% APS, 0.3µl TEMED to the bottom of the plastic tube, mix well, and let stand for 5 m...

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Abstract

The invention belongs to the fields of biochemistry, molecular biology and detection methods of nucleic acid amplification, and discloses a fast silver staining method for gel fixed at the bottom of atube for RCA visual detection. In the method, the gel containing an RCA product to be detected is fixedly arranged at the bottom of the tube, and a reagent required by silver staining is directly added into and removed from the tube to perform silver staining on the gel at the bottom of the tube to further implement semi-quantitative or qualitative analysis over an RCA sample in the gel. According to the method, a fast, convenient and visual detection technology is provided for an RCA-based detection technology, and the method has practical value and broad application prospect.

Description

Technical field [0001] The invention belongs to the field of detection methods of biochemistry, molecular biology, and nucleic acid amplification, and specifically relates to a rapid silver staining method of a tube bottom fixed gel used for RCA naked eye detection. Background technique [0002] Nucleic acid detection technology is a very widely used molecular detection method. Used for species identification, paternity identification, scientific research and many other fields. Nucleic acid amplification technology is a very commonly used nucleic acid detection technology, which achieves the enrichment of target nucleic acid and signal amplification through the amplification of specific nucleic acid sequences. [0003] Different from PCR's variable temperature nucleic acid amplification technology, rolling circle amplification (RCA) is a constant temperature amplification technology of nucleic acid, so its amplification does not require variable temperature equipment, which is mor...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6844
CPCC12Q1/6844C12Q2531/125C12Q2563/173
Inventor 赵国杰陈紫薇关一夫魏华
Owner 中国医科大学
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