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October 1, 2021
Science
Agaroses – A Solution for Every Application from GENAXXON!
Whether it's standard agarose, high-resolution agarose, or low-melting agarose – the choice of agarose in the lab depends on the size of the DNA or RNA fragments you want to separate, as well as the planned applications. Whether for blotting, PFGE, in-gel analyses, plaque assays, or immunodiffusion – Genaxxon offers the right solution for every application.
Genaxxon offers several high-resolution agaroses:
GENAXXON offers two low-melting agaroses:
Want to find the ideal agarose for your experiments and get valuable tips? Keep reading!
Which agarose is right for your application?
Genaxxon offers a wide selection of high-quality agaroses, all with low sulfate content and a low EEO (electroendosmosis) value. They are universally applicable in all gel buffer systems and are of course DNase- and RNase-free.
Still unsure which agarose is optimal for your experiment? Our chart and selection guide below will help you make the perfect choice for your application.
Agarose Gel Electrophoresis: A Practical Overview
The preparation of an agarose gel begins with dissolving agarose in an electrophoresis buffer (TAE or TBE). Upon cooling, the agarose forms a gel whose pore size directly depends on the agarose concentration. The higher the concentration, the smaller the pores, and the firmer the gel. This characteristic affects the separation of molecules – larger DNA fragments are held back more in the gel than smaller ones.
The Principle of Gel Electrophoresis
In the electric field, negatively charged DNA molecules migrate through the gel's pores toward the positive pole. Smaller molecules pass through the pores faster than larger ones, enabling size-based separation.
Visualization and Documentation
Fluorescent dyes like SafeGel replace toxic ethidium bromide to visualize DNA bands. The analysis of DNA bands is done with a DNA ladder as a reference for size and concentration.
Influencing Factors: Agarose and Buffer
In addition to agarose concentration, the charged groups of agarose and the electrophoresis buffer used can influence DNA molecule separation:
- The charged groups of agarose (pyruvate and sulfate groups) determine the electroendosmosis (EEO value)
- Negative sulfate ions are bound to the gel matrix, while cations (K+, Na+) are freely mobile. During electrophoresis, both groups move against each other, causing an unwanted braking effect. As a result, a high EEO value (e.g., high sulfate content in agarose) slows down DNA migration and causes band smearing.
- TAE Buffer: has low ion strength and low buffering capacity. It is recommended for separating larger DNA molecules (from 1kb) and when the separated DNA should be isolated afterward.
- TBE Buffer: has high ion strength and high buffering capacity. It forms sharper bands with smaller DNA molecules and is recommended for separating small DNA molecules (<1kb).
GENAXXON offers the following agaroses for DNA molecule separation:
1. High-Resolution Agaroses
- Enables separation of differences as small as 2-3 base pairs.
- Gel concentrations up to 4% are possible.
- High-resolution agaroses can be mixed with other agaroses.
- The addition of Agarose Tiny (M3046) to Agarose LE (M3044) can significantly improve separation properties and resolution.
- Agarose Tiny (M3046) can replace polyacrylamide gels.
The melting and gelation temperatures of agaroses can be influenced by chemical modifications, often reducing the number of hydrogen bonds responsible for crosslinking the agarose's twisted polysaccharide chains. The degree of modification directly impacts the melting and gelation temperatures of the agarose.
Special properties of low-melting agaroses:
- Forms gels with higher transparency compared to standard agarose.
- Due to the low melting point, ideal for quick DNA extraction from gels.
- Suitable for "in-gel" DNA processing with enzymes.
- Well-suited for DNA and RNA re-isolation from gels using a gel extraction kit.
- Suitable for digestion with β-agarase.
- Large DNA fragments can be isolated gently.
- Suitable for cell and tissue culture (M3049 – Agarose LM – low melting).
- Ideal for phage plaque assays.
1. Blotting (Southern Blot)
DNA is first digested with restriction enzymes, then separated by agarose gel electrophoresis. Alkaline treatment splits the separated DNA into single strands, which are then transferred onto a nitrocellulose membrane (blotting). Labeled RNA probes complementary to the target sequence are added. If the sequence is present on the membrane, the probe binds to it (hybridization) and can be detected based on the label.
Recommended agaroses for blotting experiments:
- M3044 – Agarose LE (Standard agarose)
- M3054 – Agarose LE Tablets
- M3047 – Agarose Tiny HT
- M3051 – Agarose Mega
2. PFGE (Pulsed-Field Gel Electrophoresis)
PFGE is a method for determining the size of chromosomal DNA fragments (30-50kb), genetic fingerprinting, bacterial genome typing, or pathogen identification. The difference from normal agarose gel electrophoresis is the use of a periodically reversed electric field, which significantly improves resolution in the 30-50kb range.
Recommended agarose for PFGE:
A plaque assay detects and quantifies infectious viruses.
For cell culture: an agarose layer containing the virus is incubated, and after incubation, the agarose is scraped off and cells are stained. The viral plaques appear as transparent spots.
For bacterial cultures: sensitive bacteria are mixed with agarose and plated, then viruses (phages) are added. After incubation, phage plaques are evaluated as transparent spots.
Recommended agarose for phage plaque assays:
Immunodiffusion is an immunochemical test for detecting antigen-antibody reactions. One popular method is the Ouchterlony test, where antigen and antibody are pipetted into pre-punched holes in an agarose-coated glass plate, and they diffuse toward each other. Precipitation lines form where antigen and antibody meet in equivalent concentrations.
Recommended agarose for immunodiffusion:
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