Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

A Rapid Silver Staining Method for RCA Amplified Products

A technique for amplified products and silver staining, which is applied in the detection of nucleic acid amplification, molecular biology, and biochemistry, and can solve the problems of inconvenient operation of changing liquids and large amount of silver staining reagents used

Active Publication Date: 2020-10-30
中国医科大学
View PDF1 Cites 0 Cited by
  • 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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • A Rapid Silver Staining Method for RCA Amplified Products
  • A Rapid Silver Staining Method for RCA Amplified Products
  • A Rapid Silver Staining Method for RCA Amplified Products

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0028] (1) Design and preparation of ordinary RCA ring formwork:

[0029] Design the template sequence of common RCA, prepare 10 μl of 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°C 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 of circular template), and perform rolling circle amplification reaction.

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

[0033] ① Preparation of tube bottom glue: Add 10µl of RCA amplification products with dif...

Embodiment 2

[0040] The present invention utilizes repressor-RCA to qualitatively identify galactose and other 5 kinds of 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 of ligation reaction system buffer (50 mM TrispH 8.0, 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 °C 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), add a sugar to carry out rolling circle amplification reaction. The final concentrations of sugars were 100 μM (galactose) and 10 mM (other sugars: fructose, fucose, glu...

Embodiment 3

[0052] Embodiment 3 The present invention detects the RCA product of different reaction times

[0053] (1) Design and preparation of ordinary RCA ring formwork:

[0054] Design the template sequence of common RCA, prepare 10 μl of 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°C 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 of circular template), and perform rolling circle amplification reactions for different times.

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

[0058] ①Preparation of tube bottom glue: add 10µl RCA amplification product, 3µl 40%ACR, 1µl 10%APS, 0.3µl TEMED to the bottom of the p...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

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 in particular relates to a rapid silver staining method for fixed gel at the bottom of a tube used for RCA naked eye detection. Background technique [0002] Nucleic acid detection technology is a widely used molecular detection method. It is used in many fields such as species identification, paternity identification, scientific research and so on. Nucleic acid amplification technology is a very commonly used nucleic acid detection technology, through the amplification of specific nucleic acid sequences to achieve the enrichment of target nucleic acids and signal amplification. [0003] Different from the temperature-changing nucleic acid amplification technology of PCR, rolling circle amplification (RCA) is a nucleic acid constant-temperature amplification technology, so its amplification does not require temperature-changin...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Patents(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
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com