Gel electrophoresis 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.

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 Cut Sites Reference Table
Restriction enzymes are a broad class of DNA-cutting enzymes that occur naturally in bacteria. Click the link to check out our dataset table containing a full list of restriction enzymes.

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:

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.

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 (TAE) 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.

Buffer Preparations and Recipes
Click the link 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.