Illustrated examples of horizontal and vertically-oriented electrophoresis gels, with outlined ladder lanes. Electrophoresis bands may vary in width, and ladders made or selected for a given procedure should match as closely as possible with expected results. If fragment sizes are entirely unknown, multiple ladders can be included for reference. Figure made with Biorender.
is a technique that separates charged molecules, like DNA and RNA
, according to size. To accurately distinguish fragment lengths of samples, DNA and RNA ladders, also termed markers, are used as the standard(s) that run concurrently with the sample. These ladders are composed of a solution of fragments of known sizes that act as molecular weight identifiers, and help to estimate the sizes of fragments in the test samples.
These ladders utilize the inherent concept of gel electrophoresis, in that molecular weight is inversely proportional to the migration of a fragment length in the gel. By aiding in distinguishing the molecular weight of fragments, base pair length may also be identified for a given set of fragments, and by proxy, the test sample.
Principles & Applications
ReadiUse™ 1 Kb Plus DNA Ladder (5 uL/well) was run on 1.2% agarose gel with 1 X TBE Buffer.
Various DNA and RNA ladders are commercially available, though similar techniques are used across the industry. One of the most commonly used methods of creating DNA and/or RNA ladders involves partial restriction digestion where the DNA or RNA molecule is only partially digested, or cut, at identified points of interest by a restriction enzyme.
The starting molecule may be composed of a vector and insert, or may be an insert without the plasmid DNA, and inserts are usually composed of concatemerized subunits (also known as a concatemer, these are DNA molecules composed of repeating copies of the same sequence) which can simplify identifying the molecular weight of subsequently formed fragments. The plasmid then undergoes further deproteinization steps for further fragmentation.
Another common technique is the creation of a ladder through partial ligation. In this method uncloned subunits of DNA are covalently joined via the activity of ligase
, an enzyme, to form concatemers. Due to the extensive ability of polymerization and ease of the experimental technique, PCR
has become a more common alternative of ladder creation compared to the previously mentioned methods. As PCR is a reliable method of rapidly producing many copies of a DNA molecule, a single desired DNA band may undergo amplification, and then be mixed with other purified PCR products at certain concentrations to form the ladder.
Additionally, multiplex PCR techniques have been used that amplify several different DNA sequences simultaneously, and PCR combined with restriction digestion has also been used to form DNA ladders. In the same way that PCR can form DNA ladders, RT-PCR
can be used to form ladders from an RNA molecule.
Restriction enzymes are a broad class of DNA-cutting enzymes that occur naturally in
bacteria. Click the button on the right to check out our dataset table containing a full list of restriction enzymes.
Visualization and Analysis
Illustration of the 3 possible conformations of plasmid DNA, which include supercoiled, linear, and circular types, in order from fastest to slowest of their typical speed of migration through the gel during electrophoresis. Figure made in Biorender.
After electrophoresis, the gel will appear with multiple “bands”, indicative of where certain sized fragments deposit within the gel. The ladder aisle may present with multiple bands, depending on the number of differently sized fragments within the ladder solvent, where a sample aisle may present with fewer bands, or commonly, one. By comparing the bands from the sample to the bands in the ladder, the size and molecular weight of fragments may be estimated.
In analyzing a gel after electrophoresis, the conformation of DNA will also affect its migration. Plasmid DNA may exist in three states, each with different migratory capabilities:
- When DNA is supercoiled, it is in its most compact state and will migrate the fastest through the gel.
- In a linear conformation, the DNA is more flexible and will migrate at a medium speed.
- In its native, relaxed, circular conformation, will migrate the slowest due to an inherent stronger resistance to the gel.
Purity of the DNA or RNA sample may also be easily analyzed upon visualization. Contaminating nucleic acids will often present as unanticipated, possibly smudgy, bands that migrate faster or slower for RNA and DNA, respectively, than the expected plasmids.
Table 1. Summary of methods for staining proteins in gel
|Coomassie Dye||5 - 25 ng||10 - 135 minutes|
- Mass spectrometry
- Western blotting
- Quick and easy staining protocol
|Silver stain||0.25 - 0.5 ng||30 - 120 minutes||
- Most robust and sensitive colorimetric method
- Protocol requires several steps
- Stain uses either glutaraldehyde or formaldehyde as an enhancer, which can cause proteins to crosslink in the gel matrix
|Zinc stain||0.25 - 0.5 ng||15 minutes|
- Mass spectrometry
- Western blotting
- Procedure stains the polyacrylamide gel and not the proteins
- No fixation steps required
- Stain can be easily removed
|Fluorescent dye||0.25 - 0.5 ng||60 minutes|
- UV/blue/green-light transilluminators
- Gel documentation systems
- Laser scanners
- Mass spectrometry
- Western blotting
- Wide linear dynamic range and sensitive
- Compatible with destaining and protein recovery methods
- Routinely used in 1D and 2D applications
PageTell™ Prestained 10 to 250 kDa Protein Ladder was run on different percentages of Tris-Glycine gels.
In electrophoresis, some general considerations must be taken. RNA and DNA ladders are not used interchangeably as RNA migrates faster than DNA throughout the gel. RNA markers can also exist in various forms including single-stranded (ssRNA), double-stranded (dsRNA), microRNA, and mRNA.
Choosing the right ladder is crucial to any successful experiment, and the number one consideration should be the expected size of the anticipated bands in the test sample. Ladders exist in a range of sizes
, commonly from 50 - 10,000 bp, and if desired multiple ladders may be used in separate aisles if the expected sample fragment sizes are unknown.
Ladders may contain either one or multiple fragment sizes, and though ladders with more bands makes estimating test sample fragments easier, the gels must run longer for a clearer interpretation of band differentiation. Ladders with multiple bands should be used if the precision of fragment identification takes priority. Conversely, if time is limited, a ladder with fewer bands may work well if only a rough estimate of band sizes is needed.
The experimental conditions, likewise, may affect ladder and sample visualization. When selecting buffers, those that contain Tris
, acetate, and EDTA
) are commonly used for ladders with larger fragments while those that contain Tris, borate, and EDTA (TBE) are typical for ladders with smaller fragments.
Though a high voltage and short run time may provide better resolution for very small fragments, too high a voltage may affect the overall resolution and mobility of most bands. The impact of an extremely high voltage can easily be seen when gels become overheated, even melted, and make the appearance of bands broader. Contrarily, too low a voltage may decrease ladder mobility and cause bands to not separate properly, forming a bleed across the ladder aisle.
Click the button on the right to see a variety of buffer preparations and recipes.
To view a list of recipes specifically for Gel Electrophoresis, select the "Gel Electrophoresis" Tab.
The gel concentration may also play a role in visualization where generally, increased gel concentration decreases fragment migration. Ladders and samples with smaller anticipated bands are more easily resolved at higher gel concentrations, and vice versa for those with larger anticipated bands.
Product Ordering Information
Table 2. ReadiUse™ DNA ladders
Table 3. Gelite™ DNA Ladders
Table 4. Nucleic acid stains for agarose and polyacrylamide gel electrophoresis
|Helixyte™ Green Nucleic Acid Gel Stain *10,000X DMSO Solution*||254 mn||Long path green filter||1 mL||17590|
|Helixyte™ Green Nucleic Acid Gel Stain *10,000X DMSO Solution*||254 mn||Long path green filter||100 µL||17604|
|Helixyte™ Gold Nucleic Acid Gel Stain *10,000X DMSO Solution*||254 mn||Long path green filter||1 mL||17595|
|Gelite™ Green Nucleic Acid Gel Staining Kit||254 nm or 300 nm||Long path green filter||1 Kit||17589|
|Gelite™ Orange Nucleic Acid Gel Staining Kit||254 nm or 300 nm||Long path green filter||1 Kit||17594|
|Gelite™ Safe DNA Gel Stain *10,000X Water Solution*||254 nm, 300 nm or 520 nm||Ethidium Bromide, Gel Star, Gel Green, Gel Red and SYBR filters||100 µL||17700|
|Gelite™ Safe DNA Gel Stain *10,000X Water Solution*||254 nm, 300 nm or 520 nm||Ethidium Bromide, Gel Star, Gel Green, Gel Red and SYBR filters||500 µL||17701|
|Gelite™ Safe DNA Gel Stain *10,000X Water Solution*||254 nm, 300 nm or 520 nm||Ethidium Bromide, Gel Star, Gel Green, Gel Red and SYBR filters||1 mL||17702|
|Gelite™ Safe DNA Gel Stain *10,000X Water Solution*||254 nm, 300 nm or 520 nm||Ethidium Bromide, Gel Star, Gel Green, Gel Red and SYBR filters||10 mL||17703|
|Gelite™ Safe DNA Gel Stain *10,000X DMSO Solution*||254 nm, 300 nm or 520 nm||Ethidium Bromide, Gel Star, Gel Green, Gel Red and SYBR filters||100 µL||17704|