Login

Forgot your password?
New User?
Remember me
banner banner

 

Agarose

AGAROSE - Form and Function

Introduction

United States Biological offers a wide range of Agarose products to be used for separation, analysis and purification of your nucleic acid samples. USBio’s Agarose and Agarose reagents are made from the highest purity biochemicals for maximum reliability.

Agarose is a neutral polysaccharide extracted from cell walls of certain Rhodophyceae algae belonging to the Gelidiaceae and Gracilariaceae families. The structure of Agarose is a linear polymer composed of alternating beta-1,3-linked D-galactose and alpha-1,4-linked 3,6-anhydro-L-galactose infrequently substituted with pyruvate, sulfate and methyl esters (Figure 1).


Figure 1. Structure of agarose polymer
Figure 1. Structure of agarose polymer.

 

The Agarose gel has a macroreticular structure which is formed by hydrogen bonds. The gel is thus thermo-reversible and it melts after heating. Agarose solutions exhibit hysteresis in the liquid-to-gel transition, that is, their gel-forming temperature is not the same as their melting temperature. Because Agarose melts easily this is an easy technique for DNA recovery after separation.


Critical Characteristics

The most critical characteristics to consider when determining the suitability and performance of an Agarose gel are: the size of DNA to be separated, the melting temperature, the gel strength, and Electroendosmosis (EEO).


1. Size of DNA to be Separated
In Agarose gel electrophoresis, the gel provides the stationary phase and the movement of the sample is achieved under an electric current. Charged particles such as DNA will migrate towards the positively charged anode in response to an electrical current across the gel. DNA has to "find its way" through the Agarose. Smaller fragments move more rapidly than larger fragments. Increasing the percent of the gel decreases the size of the pores.

The correct percent Agarose gel is dependent on the size of the fragment that will be tested. Plasmid DNA preparations that are 5kb to 7kb resolve well on a 1% gel. Large PCR fragments that are similar in size to plasmid DNA could also be resolved on a 1% percent gel; however, small PCR fragments that require a smaller pore size for better resolution require a higher percent gel. General guidelines for mixing the correct percent gel are provided in Table 1.


Table 1

Percent Agarose gel (w/v)

DNA size resolution (kb=1000bp)

0.5% 1kb to 30kb
0.7% 80bp to 12kb
1.0% 500bp to 10kb
1.2% 400bp to 7kb
1.5% 200bp to 3kb
2.0% 50bp to 2kb
 

 

The majority of US Biological Agarose products follow the table above for DNA size resolution. A few of our products are prepared specifically for much smaller primer size fragments in the range of 10-2000 base pairs. These Agarose products listed below follow the gel preparation guidelines found in Table 2.


Catalog #

Product Name

A1000 Agarose DNA Fragments 10-500bp
A1005 Agarose DNA Fragments 50-1000bp
A1010 Agarose DNA Fragments 150-1500bp
A1012 Agarose DNA Fragments 50-2000bp (3:1 Blend)

 

Table 2

Percent Agarose gel (w/v)

DNA size Molecular Weight

1.8% 500-1000bp
3.0% 150-600bp
4.5% 50-350bp

 

2. Melting/Gel Temperature
Since the purification of DNA from Agarose by standard methods such as electroelution is inconvenient and can result in low yields, a revised method was needed to allow DNA embedded in Agarose to be used effectively without lengthy, inefficient purification steps. Some naturally occurring Agaroses were known to exhibit lower gelling and melting temperatures due to a higher methoxyl content. It was proposed that methylation of agarose under certain conditions (thereby increasing the methoxyl content), could change the melting/gelling temperatures. This proved to be the case; melting and gelling temperatures are changed drastically by methylation as well as other processes such as alkylation and hydroxyalkylation.

A1020 The low melting point also ensures that the Agarose will be in a liquid state where in-gel manipulations can be performed without prior purification of the DNA from the gel slice.

 

Catalog #

Product Name

A1020 Agarose Low Melting

 

3. Gel Strength
Gel strength is particularly important when gels must be handled or blotted after electrophoresis. One Agarose modification produces a re-arrangement of the double helical structures in the Agarose molecules and leads to increased strength of the gel formed. Low concentration gels required for pulsed-field gel electrophoresis in particular, can be very fragile when prepared with standard grade Agarose.

A1030, High Gel Strength Agarose combines very high gel strength with low EEO and is a good choice for high speed, high temperature pulsed field gel electrophoresis applications.

 

Catalog #

Product Name

A1025 Agarose High Gel Temp
A1030 Agarose High Gel Strength

 

4. Electroendosmosis (EEO)
Electroendosmosis (EEO) is one of the most important characteristics to consider when choosing an Agarose product to meet your electrophoretic needs. EEO is the movement of non-charged molecules through a medium toward the cathode during electrophoresis. If Agarose is the medium, ions such as ester sulfate and pyruvate groups impart a net negative charge to the Agarose. Though the gel itself cannot move, counter-ions and water associated with the sulfate and pyruvate groups move toward the cathode. As water migrates with the counter-ions, neutral molecules that normally would not migrate are pulled along with the water. This leads to swelling and distortion of the gel. In addition, the movement of water in the gel is in a direction opposite to the movement of the proteins, which leads to unwanted broadening of the protein band. This movement of molecules towards the cathode is known to slow the separation of DNA; so the lower the EEO, the faster DNA will migrate. Additionally, lower EEO helps improve the resolution of DNA and RNA as their migration is determined only by their size, not by their charge. The use of High EEO Agarose, although generally undesirable, is sometimes unavoidable when separating very high molecular weight proteins. Researchers often try to find the lowest EEO Agarose product that meets their needs.


United States Biological offers Agarose with a variety of EEO characteristics.

Catalog #

Product Name

A1015 Agarose DNA Fragments 10-500bp
A1016 Agarose DNA Fragments 50-1000bp
A1035 Agarose DNA Fragments 150-1500bp
A1040 Agarose DNA Fragments 50-2000bp (3:1 Blend)
A1045 Agarose DNA Fragments 150-1500bp

 

A1015 and A1016 are the “go-to” Agarose products for a wide array of applications. These Agarose gels offer enhanced resolution ensuring high recovery of biologically active DNA, high cloning efficiency and consistent reproducibility.

A1035 and A1040 are suitable for general use Agarose gels.

A1045, Extra High EEO Agarose is especially suitable for large molecule applications due to the exceptionally high cathode migration possible.

US Biological application references:

  1. Stewart, C.R. et al., (2009) J. Bacteriol. 191: 1537-1546. (A1020)
  2. Cole, K. S. (2012) “Ultra-Stable Protein-Polymer Bioconjugates”, http://digitalcommons.uconn.edu/cgi/viewcontent.cgi?article=1410&context=gs_theses (A1015)

 

Agarose-Related Reagents

These reagents are frequently used together with Agarose in a variety of applications.


Catalog #

Product Name

Sizes Availables

C3010 Cesium Chloride 500g, 1Kg
D3150 Denhardt’s Solution 50X 10ml, 25ml
D3880 DNA, Calf Thymus (Deoxyribonucleic acid) 100mg, 1g
D3950 DNA, Salmon Testes (Deoxyribonucleic acid) 1g, 5g
D3951 DNA, Salmon Sperm (Deoxyribonucleic acid) 5x1ml
D8070 Dithiothreitol (DTT) 10g-100g
E2210 EDTA Disodium Salt Dihydrate 500g-10Kg
G8145 Glycerol (Glycerine) 1L-20L
G9010 Guanidine Hydrochloride 500g-10Kg
G9015 Guanidine Hydrochloride 6M Solution 500ml
P9100 Proteinase K 100mg-1g
P9101 Proteinase K Solution 20mg/ml 1ml-50ml
R2011 Ribonuclease A, Bovine Pancreas (RNase A) 100mg-5g
S5010-01 Sodium Dodecyl Sulfate (SDS) 20% Solution 100ml, 500ml
T8600 Tris Base Ultrapure 1-25kg
T8600-05 Tris Acetate 100g, 500g, 1Kg
T8601 Tris Base USP 1kg, 5kg
T8650 Tris Hydrochloride (Tris HCl) 1-25kg
U2010 Urea 1Kg, 2.5Kg, 5Kg
W0900 Water, Sterile, Nuclease Free 500ml, 1L, 3L