What Is the Invitrogen 100 bp Ladder?
Examine the Invitrogen 100 bp ladder, a molecular biology standard used as a reference scale to estimate DNA fragment size in gel electrophoresis.
Determining the size of DNA molecules is a common task in molecular biology. Scientists use a tool known as a DNA ladder to estimate the size of unknown DNA fragments. The Invitrogen 100 bp Ladder is a commercial product that serves as a molecular-weight standard in laboratories for sizing double-stranded DNA.
Defining the Invitrogen 100 bp Ladder
A DNA ladder is a solution containing a mixture of DNA molecules of known sizes. When used in gel electrophoresis, these fragments separate to form a series of bands resembling the rungs of a ladder. The “100 bp” designation in the Invitrogen 100 bp Ladder indicates the mixture contains DNA fragments that start at 100 base pairs (bp) and increase in 100 bp increments. A base pair is the basic unit of the DNA double helix.
This ladder is produced by Invitrogen and provides a scale of predefined sizes against which unknown DNA fragments can be compared. The fragments range from 100 base pairs up to 2,000 base pairs, allowing for the sizing of DNA across a broad spectrum.
The product’s purpose is to serve as a size standard, not as a part of the experiment’s reaction. It is a measurement tool that runs alongside experimental samples, providing reliable reference points. This allows researchers to confirm the size of their DNA from a polymerase chain reaction (PCR) or restriction enzyme digest.
Key Characteristics and Composition
The Invitrogen 100 bp DNA Ladder is designed for sizing and approximating the quantity of double-stranded DNA. A preparation consists of 13 individual, purified DNA fragments that range in size from 100 to 2,000 base pairs. The specific bands are:
- 100 bp
- 200 bp
- 300 bp
- 400 bp
- 500 bp
- 600 bp
- 700 bp
- 800 bp
- 900 bp
- 1000 bp
- 1200 bp
- 1500 bp
- 2000 bp
To aid in identification, certain fragments are intentionally made brighter. In this ladder, the 600 bp and 1,500 bp bands serve as enhanced reference points. This increased intensity helps with orientation when analyzing a gel image, making it easier to identify the other bands.
The product is supplied in a ready-to-use format, premixed with a loading buffer like BlueJuice Gel Loading Buffer. This buffer contains a dye to track DNA migration and a density agent to ensure the ladder settles into the gel’s well. For stability, the ladder is stored at –20°C.
Application in DNA Fragment Sizing
The primary application for the Invitrogen 100 bp Ladder is as a size marker during agarose gel electrophoresis, a technique that separates DNA fragments by size. The process uses a porous agarose gel submerged in a buffer. The ladder is loaded into an indentation, or well, at one end of the gel, with experimental samples loaded into adjacent wells.
An electric current is then applied across the gel. DNA has a net negative charge, so the fragments move toward the positive electrode. The gel matrix acts as a sieve; smaller fragments navigate the pores more easily and travel farther, while larger fragments move more slowly.
The ladder separates into a predictable pattern of bands, creating the reference scale for size estimation. By running the ladder next to unknown samples, researchers can compare the migration distance of their sample bands to the ladder bands. This comparison allows for the accurate sizing of the experimental DNA fragments.
Interpreting Gel Electrophoresis Results
After the gel run is complete, it is stained with a DNA-binding fluorescent dye and visualized under UV or blue light. This process reveals the separated DNA fragments as distinct bands. The lane containing the ladder will show a series of bands corresponding to the known fragment sizes, from the smallest band that traveled farthest to the largest that traveled the shortest.
To determine the size of an unknown DNA fragment, a researcher locates the sample’s band and compares its position to the ladder’s bands in the adjacent lane. For instance, if a sample band aligns horizontally with the 500 bp band of the ladder, its size is estimated to be approximately 500 base pairs.
The quality of the bands also provides information. Sharp, well-defined bands in the ladder and sample lanes indicate a successful experiment. Issues like smearing can suggest DNA degradation, while multiple unexpected bands might point to contamination or non-specific products.