Name: Agarose Gel Electrophoresis of DNA Amplicons Post PCR: A Method to Analyze Products of Multiplex PCR and Evaluate PCR Reaction Success
Uploaded: 2023-04-30T15:00:07
Description: Source: Gulla, S. et al. Multi-locus Variable-number Tandem-repeat Analysis of the Fish-pathogenic Bacterium Yersinia ruckeri by Multiplex PCR and ...

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1. PCR Amplicon Confirmation by Gel Electrophoresis
<ol>
	<li>According to the manufacturer&#39;s recommendations, prepare a volume of 1.5% (w/v) agarose gel in 1x tris-borate-EDTA (TBE) buffer appropriate for the number of PCR reactions to be tested. Prior to casting, add 5 &#181;L of fluorescent nucleic acid dye per 50 &#181;L of gel solution and mix. Use trays and combs as appropriate for casting, leaving an appropriate number of wells free for DNA reference ladders.</li>
	<li>After setting, submerg...

agarose gel electrophoresis of dna amplicons post pcr: a method to analyze products of multiplex pcr and evaluate pcr reaction success

Source: Gulla, S. et al. <a href="https://www.jove.com/v/59455">Multi-locus Variable-number Tandem-repeat Analysis of the Fish-pathogenic Bacterium Yersinia ruckeri by Multiplex PCR and Capillary Electrophoresis</a>. J. Vis. Exp. (2019)
In this video, we demonstrate the separation of bacterial PCR-amplified DNA using agarose gel electrophoresis. Agarose gel functions as a molecular sieve, enabling the negatively-charged DNA to migrate based upon their size under an applied electric field.

<img alt="Figure 1" src="/files/ftp_upload/21112/21112_Figure1.jpg" width="700px" /> 
Figure 1: Gel electrophoresis verifying the presence of multiple PCR products. The image confirms the presence of multiple PCR amplicons in all 12 lanes containing samples, with the first lane representing the DNA ladder used. The sizes of selected ladder fragments have been indicated, as have the PCR assay and strain affiliation of each lane.

Agarose Gel Electrophoresis of DNA Amplicons Post PCR: A Method to Analyze Products of Multiplex PCR and Evaluate PCR Reaction Success

Source: Gulla, S. et al. Multi-locus Variable-number Tandem-repeat Analysis of the Fish-pathogenic Bacterium Yersinia ruckeri by Multiplex PCR and ...

To analyze DNA amplicons - amplified DNA fragments of specific length - resulting from multiplex polymerase chain reaction, PCR, using agarose gel electrophoresis, assemble a casting tray with a comb to create wells for sample loading.
Pour an appropriate concentration of molten agarose, a linear polysaccharide, dissolved in electrophoresis buffer, supplemented with a fluorescent DNA dye, onto the casting tray and allow to solidify.
The molten agarose polymerizes, forming a three-dimensional gel with microscopic pores.
Place the casted gel in an electrophoresis tank, with the comb-side oriented near the negative cathode. The tank contains the electrophoresis buffer used in gel preparation to maintain optimal conductivity. Remove the comb.
Transfer the PCR product sample, mixed with loading dye solution to monitor sample migration, into the wells. Load one well with DNA ladders of defined length. Initiate electrophoresis.
DNA amplicons, with evenly spaced negative phosphate groups, migrate from the wells into the gel towards the positive anode and get separated based on their length due to high mobility of shorter amplicons than larger ones. Simultaneously, the positive fluorescent DNA dye in the gel migrates in the reverse direction and combines with DNA amplicons, intercalating between the bases.
Post electrophoresis, image the gel using ultraviolet light. For a successful PCR, the dye-intercalated DNA amplicons appear as discrete, fluorescent bands closest to the ladder of expected amplicon length. The amplicon yield can be estimated through fluorescence intensity.

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Research

JoVE Encyclopedia of Experiments

Biological Techniques

Electrophoresis Techniques

Real-time Tracking of DNA Fragment Separation by Smartphone

Slab gel electrophoresis (SGE) is the most common method for the separation of DNA fragments; thus, it is broadly applied to the field of biology and others. However, the traditional SGE protocol is quite tedious, and the experiment takes a long time. Moreover, the chemical consumption in SGE experiments is very high. This work proposes a simple method for the separation of DNA fragments based on an SGE chip. The chip is made by an engraving machine. Two plastic sheets are used for the excitation and emission wavelengths of the optical signal. The fluorescence signal of the DNA bands is collected by smartphone. To validate this method, 50, 100, and 1,000 bp DNA ladders were separated. The results demonstrate that a DNA ladder smaller than 5,000 bp can be resolved within 12 min and with high resolution when using this method, indicating that it is an ideal substitute for the traditional SGE method.

Traditional slab gel electrophoresis (SGE) experiments require a complicated apparatus and high chemical consumption. This work presents a protocol that describes a low-cost method to separate DNA fragments within a short timeframe.

Slab gel electrophoresis (SGE) is the most common method for the separation of DNA fragments; thus, it is broadly applied to the field of biology and ...

JoVE Journal

Biochemistry

DNA Electrophoresis Using Thiazole Orange Instead of Ethidium Bromide or Alternative Dyes

DNA gel electrophoresis using agarose is a common tool in molecular biology laboratories, allowing separation of DNA fragments by size. After separation, DNA is visualized by staining. This article demonstrates how to use thiazole orange to stain DNA. Thiazole orange compares favorably to common staining methods, in that it is sensitive, inexpensive, excitable with UV or blue light (to prevent sample damage), and safer than ethidium bromide. Labs already equipped to run DNA electrophoresis experiments using ethidium bromide can generally switch dyes with no additional changes to existing protocols, using UV light for detection. Blue-light detection to avoid sample damage can additionally be achieved with a blue-light source and emission filter. Labs already equipped for blue-light detection can simply switch dyes with no additional changes to existing protocols.

Here, we present a protocol to use thiazole orange for the detection of DNA in gel electrophoresis experiments. The use of thiazole orange allows elimination of ethidium bromide, and fluorescence detection can be achieved with either UV or blue light.

DNA gel electrophoresis using agarose is a common tool in molecular biology laboratories, allowing separation of DNA fragments by size. After separation, ...

Mucin Agarose Gel Electrophoresis: Western Blotting for High-molecular-weight Glycoproteins

Mucins, the heavily-glycosylated proteins lining mucosal surfaces, have evolved as a key component of innate defense by protecting the epithelium against invading pathogens. The main role of these macromolecules is to facilitate particle trapping and clearance while promoting lubrication of the mucosa. During protein synthesis, mucins undergo intense O-glycosylation and multimerization, which dramatically increase the mass and size of these molecules. These post-translational modifications are critical for the viscoelastic properties of mucus. As a result of the complex biochemical and biophysical nature of these molecules, working with mucins provides many challenges that cannot be overcome by conventional protein analysis methods. For instance, their high-molecular-weight prevents electrophoretic migration via regular polyacrylamide gels and their sticky nature causes adhesion to experimental tubing. However, investigating the role of mucins in health (e.g., maintaining mucosal integrity) and disease (e.g., hyperconcentration, mucostasis, cancer) has recently gained interest and mucins are being investigated as a therapeutic target. A better understanding of the production and function of mucin macromolecules may lead to novel pharmaceutical approaches, e.g., inhibitors of mucin granule exocytosis and/or mucolytic agents. Therefore, consistent and reliable protocols to investigate mucin biology are critical for scientific advancement. Here, we describe conventional methods to separate mucin macromolecules by electrophoresis using an agarose gel, transfer protein into nitrocellulose membrane, and detect signal with mucin-specific antibodies as well as infrared fluorescent gel reader. These techniques are widely applicable to determine mucin quantitation, multimerization and to test the effects of pharmacological compounds on mucins.

Mucins are high-molecular-weight glycoconjugates, with size ranging from 0.2 to 200 megadalton (MDa). As a result of their size, mucins do not penetrate conventional polyacrylamide gels and require larger pores for separation. We provide a detailed protocol for mucin agarose gel electrophoresis to assess relative quantification and study polymer assembly.


Mucins, the heavily-glycosylated proteins lining mucosal surfaces, have evolved as a key component of innate defense by protecting the epithelium against ...

Agarose Gel Electrophoresis of DNA Amplicons Post PCR: A Method to Analyze Products of Multiplex PCR and Evaluate PCR Reaction Success

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