Horizontal Electrophoresis

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Overview

This section provides an overview of horizontal electrophoresis, gel boxes, running buffers, and agarose types, as well as discusses some factors affecting resolution of DNA fragments and suggests appropriate agarose types and concentration for different sizes of DNA molecules. Pulsed field gel electrophoresis is discussed in a separate section.

Electrophoresis Gel Boxes

Gel boxes are available in different sizes to accommodate different needs. Mini formats are usually used for quick separations and a small number of samples; large formats are ideal for restriction fragment length polymorphism (RFLP), Southern blotting, and PCR sample screening. Bio-Rad offers multiple choices of gel boxes called Sub-cell® systems to fit different applications.

Many users cast their own gels; in fact, gels can be cast either directly in the gel box, using a gel caster, or in a UV tray on which the openings are closed off by adhesive tape. A large selection of tools, including combs with different numbers of wells and different well thicknesses, or different sized UV trays and gel casters, are available for simplifying gel casting.

For convenience, many users purchase precast gels, which are available in different sizes and gel percentages and are prepared either with Tris-acetate-EDTA (TAE) or Tris-borate EDTA (TBE) buffer. Bio-Rad offers numerous precast gels and ReadyAgarose™ gels for your convenience.

Bio-Rad's Sub-Cell® System Selection Guide

  Mini-Sub® Cell GT* Wide Mini-Sub® Cell GT** Sub-Cell® GT Sub-Cell® Model 96 Sub-Cell® Model 192
Cell size
(W x L x H)
9.2 x 25.5 x 5.6 cm 17.8 x 25.5 x 6.8 cm 18 x 40.5 x 9.4 cm 29 x 30 x 9 cm 29 x 40 x 9 cm
Gel tray sizes
(OD)
(W x L)
7 x 7 cm
7 x 10 cm
15 x 7 cm
15 x 10 cm
15 x 10 cm
15 x 15 cm
15 x 20 cm
15 x 25 cm
25 x 10 cm
25 x 15 cm
25 x 10 cm
25 x 15 cm
25 x 20 cm
25 x 25 cm
ReadyAgarose gels accommodated Yes Yes No No No
Sample throughput 8–30*** 10–60*** 1–120† 24–96*** 24–192†
Base buffer volume ~270 ml ~650 ml ~1 L ~2 L ~3 L
Buffer recirculation No No No Yes Yes
Bromophenol blue migration ~4.5 cm/hr
(at 75 V)
~4.5 cm/hr
(at 75 V)
~3.0 cm/hr
(at 75 V)
~6.2 cm/hr
(at 200 V)
~5.2 cm/hr
(at 200 V)

* Mini ReadySub-Cell GT is a Mini-Sub cell GT dedicated to running ReadyAgarose precast gels, gel size 7 x 10 cm; sample throughput is 8, 12, or 2 x 8. This cell does not include casting gates, tray, or combs.
** Wide mini ReadySub-Cell GT is a wide Mini-Sub cell GT dedicated to running ReadyAgarose precast gels, gel size 15 x 10 cm; sample throughput is 20, 32, 2 x 32, or 4 x 26. This cell does not include casting gates, tray, or combs.
*** Sample throughput value assumes 1–2 combs per gel.
† Sample throughput value assumes 1–4 combs per gel.

 

Optimal Resolution Range of ReadyAgarose™ Gels

Gel Percentage Mini Gels
8-well, 12-well
Wide Mini Gels
20-well, 32-well, 2 x 32-well
ReadyAgarose 96 Plus Gels
4 x 26-well
1% 200–10,000 bp 200–10,000 bp 200–10,000 bp
3%* 20–2,000 bp 20–2,000 bp 20–2,000 bp

* A 3% ReadyAgarose gel is easier to handle and gives the same performance as a standard 4% gel.

 

Handcast Agarose Gels
Handcasting offers options for a variety of gel sizes. Two accessories are available for handcasting gels within the Sub-Cell family of electrophoresis cells. Gel casters offer the flexibility of accommodating several different-sized UV transparent (UVTP) gel trays and are available for the entire Sub-Cell product line. Casting gates offer the convenience of casting directly in the electrophoresis cells and are available in one specific size for each cell (see Casting Guide below).

Casting Guide

Electrophoresis Cell Tray Size Casting Gates Gel Caster Electrophoresis Cell Tray Size Casting Gates Gel Caster
Mini-Sub cell GT 7 x 7 cm
7 x 10 cm

Sub-Cell Model 96 25 x 10 cm
25 x 15 cm

Wide Mini-Sub cell GT 15 x 7 cm
15 x 10 cm

Sub-Cell Model 192 25 x 10 cm
25 x 15 cm


Sub-Cell GT 15 x 10 cm

  25 x 20 cm    
  15 x 20 cm
15 x 25 cm
 
       

Hand Casting Options for a Variety of Gel Sizes. Two accessories are available for hand casting with the Sub-Cell family of electrophoresis cells: gel casters and casting gates. Gel casters offer the flexibility of accommodating several different-sized gel trays. These are available for the entire line of Sub-Cell systems. Casting gates, available in specific sizes for each cell, offer the convenience of casting directly in the Sub-Cell cells.

Electrophoresis Running Buffers

An electrophoresis buffer conducts an electric current across the electrophoresis chamber; the most commonly used buffers for DNA electrophoresis are TAE or TBE. For full recipes see table below.

Linear double stranded DNA is separated faster in TAE buffer; however, this buffer has a lower buffering capacity than TBE because the borate in the TBE buffer acts as an enzyme inhibitor and could negatively impact downstream applications. Therefore, TAE is the preferred buffer if the DNA will be used for cloning or ligation.

Electrophoresis Buffer Selection Guide

Buffer 1x Formulation Applications
Protein Electrophoresis    
10x Tris/glycine/SDS 25 mM Tris, 192 mM glycine,
0.1% SDS, pH 8.3
General SDS-PAGE
10x Tris/glycine 25 mM Tris, 192 mM glycine,
pH 8.3
Native PAGE
10x Tris/Tricine/SDS 100 mM Tris, 100 mM tricine,
0.1% SDS, pH 8.3
Peptide SDS-PAGE
10x IEF anode buffer 7 mM phosphoric acid Analytical isoelectric focusing
10x IEF cathode buffer 20 mM lysine, 20 mM arginine Analytical isoelectric focusing
10x zymogram 2.5%
renaturation buffer
Triton X-100 Protease analysis;renatures enzymes after electrophoresis
10x zymogram
development buffer
50 mM Tris-HCl, pH 7.5,
200 mM NaCl, 5 mM CaCl2,
0.02% Brij-35
Protease analysis; activates enzymes after electrophoresis
Nucleic Acid Electrophoresis    
10x TBE 89 mM Tris, 89 mM boric acid, 2 mM EDTA, pH 8.3 Nucleic acid electrophoresis/sequencing;
polyacrylamide or agarose gels
10x TBE
extended range
130 mM Tris, 45 mM boric acid, 2.5 mM EDTA Nucleic acid electrophoresis/sequencing;
polyacrylamide or agarose gels; extends the buffer capacity for longer DNA sequencing runs
50x TAE 40 mM Tris, 20 mM acetic acid, 1 mM EDTA, pH 8.0 Nucleic acid electrophoresis;
polyacrylamide or agarose gels

Blotting Buffer Selection Guide

  1x Formulation Applications
Transfer Buffers*    
10x Tris/glycine 25 mM Tris, 192 mM glycine, pH 8.3 Western blotting
10x Tris/CAPS Anode buffer: 60 mM Tris,
40 mM CAPS, 15% methanol, pH 9.6
Cathode buffer: 60 mM Tris,
40 mM CAPS, 0.1% (w/v) SDS, pH 9.6
A discontinuous buffer system that increases transfer efficiency in semi-dry applications
20x SSC 150 mM sodium chloride,
15 mM sodium citrate, pH 7.0
Capillary transfer of agarose gels
Processing Buffers    
10x PBS 10 mM sodium phosphate,
150 mM NaCl, pH 7.4
Western blotting wash solution
10x TBS 20 mM Tris,500 mM NaCl, pH 7.4
150 mM NaCl, 1% (w/v) casein, pH 7.4
Western blotting wash solution
1x PBS with 1% casein 10 mM sodium phosphate,
150 mM NaCl, 1% (w/v) casein, pH 7.4
Western blotting blocking buffer (casein blockers recommended for all applications, including those with biotin-avidin complexes)
1x TBS with 1% casein 20 mM Tris, 500 mM NaCl
containing 1% (w/v) casein, pH 7.4
Western blotting blocking buffer (casein blockers recommended for all applications, including those with biotin-avidin complexes)
20x SSC 150 mM sodium chloride,
15 mM sodium citrate, pH 7.0
Northern and Southern blotting prehybridization and hybridization solutions

* These buffers can be used for all gel types and formulations.

Agarose Types

A variety of agarose powders are available. When selecting the type of agarose to use, the most important factor to consider is the size of the fragments being separated. In addition, considering the percentage of agarose in the gel may better resolve the fragments of interest and produce a gel that will yield a publication-quality image. Higher percentage gels resolve smaller DNA fragments, whereas lower percentage gels resolve larger fragments. Each type of agarose in Bio-Rad's Certified™ Agarose product line is genetic quality tested (GQT) and can be used for routine separations and downstream molecular biology applications.

Analytical Separation

  Molecular Biology Agarose PCR Agarose Low Range Ultra Agarose Low-Melt Agarose PCR Low-Melt Agarose Megabase Agarose Pulsed Field Agarose
>1,000 bp        
<1,000 bp        
10–200 bp          
1 kb–2 Mb        
1 kb–5 Mb          
Preparative Separation
Quantum Prep methods    
Low-melt methods        
Agarose methods        
In-gel applications        
Other Applications
Postpreparative enzymatic treatments  
Tissue/cell culture          
Pulsed field sample preparation        
Blotting    

Nucleic Acids Blotting

Nucleic Acids Stains and Tracking Dyes
Nucleic acid molecules are colorless and a tracking dye such as bromophenol blue or xylene cyanol is generally used to track their movement in the gel. After electrophoresis, nucleic acids are stained. The fluorescent stain ethidium bromide is the most commonly used, but other stains are available. After staining, the gel is visualized using a gel imager.

Routine Separations
Certified molecular biology agarose is recommended as a general-purpose agarose for routine separations of ~500 bp to 20 kb DNA fragments. This agarose offers rapid migration rates, easy-to-manipulate gels, and displays high strength even at low agarose percentages.

Influence of gel percentage on optimal DNA fragment resolution

Gel Percentage Optimal Resolution Range
0.75% 500 bp–10 kb
1.00% 300 bp–9 kb
1.25% 100 bp–8 kb

Separations of Small Fragments
For high resolution separations of small fragments such as PCR amplified fragments, Certified PCR agarose is recommended. This agarose separates DNA fragments from 20 bp to 1000 bp. The standard gelling temperature makes it easy to prepare, and the gels are easy to manipulate and remain flexible even at high gel percentages. This agarose offers a higher resolution of small fragments than the Certified molecular biology agarose.

 

Influence of gel percentage on optimal fragment resolution

Gel Percentage Optimal Resolution Range
2% 100 bp–2.5 kb
3% 40 bp–2 kb
4% 20 bp–1 kb

 

Separations of Very Small Fragments
For the separation of extremely small PCR fragments and primers (~10 to 200 bp), Certified low range ultra agarose is recommended. This agarose provides exceptionally higher resolution than standard gels at lower concentrations, allowing visualization of differences of ~5 bp in the 10–100 bp range. In this low range, the low range ultra agarose provides higher resolution than the PCR agarose.

Influence of gel percentage on optimal fragment resolution

Gel Percentage Optimal Resolution Range
2% 10 bp–1 kb
3% 10 bp–400 bp

Low-Melt Applications for Fragments <1000 bp
For applications such as cloning, in-gel applications, or DNA and RNA fragment recovery, Certified PCR low-melt agarose is recommended. This agarose has high-sieving capacity and offers excellent resolution of fragments <1000 bp in low-melt applications.

Influence of gel percentage on optimal fragment resolution

Gel Percentage Optimal Resolution Range
2% 40 bp–2 kb
3% 20 bp–1 kb
4% 10 bp–600 bp

Low-Melt Applications for Fragments >1000 bp
For separations of molecules greater than 1000 bp for clamped homogeneous electrical field (CHEF) separations, recovery of a larger fragment, or sealing IPG strips in 2-D SDS-PAGE gels, Certified low-melt agarose is recommended.

Influence of gel percentage on optimal fragment resolution

Gel Percentage Optimal Resolution Range
1.00% 100 bp–5 kb
1.25% 100 bp–3 kb

Separation of Large DNA for Pulsed Field Gel Electrophoresis (PFGE)
For analytical separation of large DNA fragments requiring PFGE, pulsed field Certified agarose is recommended and an optimal separation range of 1 kb to 2 Mb is available as a preset, selectable method of the CHEF Mapper® XA system. This agarose can also be used for blotting.

Faster Migration Rate for CHEF and FIGE
For CHEF applications, PFGE, separation of megabase DNA, or other large fragment analysis, Certified megabase agarose is suggested due to its high exclusion limit, high electrophoretic mobility, and very high gel strength. The gels are easy to handle even at 0.3%, offer short run times, and have a separation range between 1 kb and 5 Mb.

Certified megabase agarose has superior electrophoretic mobility, faster migration rates, faster running times, and better separation of megabase fragments than the pulsed field Certified agarose.

Related Content

Literature
Number Description Download
2414 The Little Book of Standards, Rev D Click to download
2121 User Guide to Nucleic Acid Standards Bulletin, Rev D Click to download
Number Description Options
6180 Hand Casting Options for a Variety of Gel Sizes Click to download
6206 Agarose Gel Preparation for DNA Separation Click to download
6230 Nucleic Acid Electrophoresis Click to download
6181 Sub-Cell Selection Guide Click to download
6182 Mini-Sub® Cell GT Complete Systems Click to download
6183 Mini-Sub® Cell Comb Selection Guide Click to download
6184 Wide Mini-Sub® Cell GT Complete Systems Click to download
6185 Wide Mini-Sub® Cell Comb Selection Guide Click to download
6186 Sub-Cell® GT Complete Systems Click to download
6187 Sub-Cell® GT Comb Selection Guide Click to download
6188 Sub-Cell® Model 96 Complete Systems Click to download
6189 Sub-Cell® Model 96 Comb Selection Guide Click to download
6190 Sub-Cell® Model 192 Complete Systems Click to download
6191 Sub-Cell® Model 192 Comb Selection Guide Click to download
6192 Certified Agarose Selection Guide Click to download
6193 Imaging System Selection Guide Click to download
6205 Electrophoresis Buffers, Bulletin 6205 Click to download
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