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.
Page Contents
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 |
|
|
Electrophoresis Cell | Tray Size |
|
|
|---|---|---|---|---|---|---|---|
| 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.
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.
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.
| Molecular Biology Agarose | PCR Agarose | Low Range Ultra Agarose | Megabase Agarose | Low-Melt Agarose | PCR Low-Melt Agarose | Pulsed Field Agarose | |
|---|---|---|---|---|---|---|---|
| Analytical Separation | |||||||
| ≥1,000 bp | • | • | |||||
| ≤1,000 bp | • | • | |||||
| 10–800 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 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.
Documents
TEST
| 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 |