Analyzing Dna by Gel Electrophoresis Allows Researchers to

Analyzing Dna by Gel Electrophoresis Allows Researchers to

What is gel electrophoresis?

  • Gel electrophoresis is a technique unremarkably used in laboratories to separate charged molecules like DNA, RNA and proteins according to their size.
  • Charged molecules move through a gel when an electrical electric current is passed beyond it.
  • An electric electric current is practical beyond the gel so that one terminate of the gel has a positive charge and the other end has a negative charge.
  • The motion of charged molecules is called migration. Molecules migrate towards the opposite charge. A molecule with a negative charge will therefore exist pulled towards the positive cease (opposites attract!).
  • The gel consists of a permeable matrix, a bit like a sieve, through which molecules tin can travel when an electric current is passed beyond information technology.
  • Smaller molecules migrate through the gel more quickly and therefore travel farther than larger fragments that drift more slowly and therefore will travel a shorter distance. As a result the molecules are separated by size.

Gel electrophoresis and Dna

  • Electrophoresis enables you lot to distinguish Dna fragments of dissimilar lengths.
  • Dna is negatively charged, therefore, when an electric current is applied to the gel, DNA will migrate towards the positively charged electrode.
  • Shorter strands of Dna move more quickly through the gel than longer strands resulting in the fragments beingness arranged in order of size.
  • The use of dyes, fluorescent tags or radioactive labels enables the Deoxyribonucleic acid on the gel to be seen after they have been separated. They volition appear as bands on the gel.
  • A Deoxyribonucleic acid marker with fragments of known lengths is usually run through the gel at the same time as the samples.
  • By comparing the bands of the Dna samples with those from the Deoxyribonucleic acid marker, y’all can work out the gauge length of the DNA fragments in the samples.
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How is gel electrophoresis carried out?

Preparing the gel

  • Agarose gels are typically used to visualise fragments of DNA. The concentration of agarose used to make the gel depends on the size of the DNA fragments you lot are working with.
  • The higher the agarose concentration, the denser the matrix and vice versa. Smaller fragments of Dna are separated on college concentrations of agarose whilst larger molecules require a lower concentration of agarose.
  • To brand a gel, agarose powder is mixed with an electrophoresis buffer and heated to a high temperature until all of the agarose powder has melted.
  • The molten gel is and so poured into a gel casting tray and a “comb” is placed at i end to brand wells for the sample to be pipetted into.
  • Once the gel has cooled and solidified (it volition now exist opaque rather than clear) the rummage is removed.
  • Many people now use pre-made gels.
  • The gel is and then placed into an electrophoresis tank and electrophoresis buffer is poured into the tank until the surface of the gel is covered. The buffer conducts the electrical current. The type of buffer used depends on the approximate size of the DNA fragments in the sample.

Preparing the DNA for electrophoresis

  • A dye is added to the sample of Dna prior to electrophoresis to increase the viscosity of the sample which volition forestall it from floating out of the wells then that the migration of the sample through the gel tin can be seen.
  • A Dna marker (besides known as a size standard or a Dna ladder) is loaded into the first well of the gel. The fragments in the marking are of a known length so can be used to assist gauge the size of the fragments in the samples.
  • The prepared DNA samples are then pipetted into the remaining wells of the gel.
  • When this is done the lid is placed on the electrophoresis tank making sure that the orientation of the gel and positive and negative electrodes is correct (we want the DNA to migrate beyond the gel to the positive cease).
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Separating the fragments

  • The electrical current is and then turned on so that the negatively charged Deoxyribonucleic acid moves through the gel towards the positive side of the gel.
  • Shorter lengths of DNA move faster than longer lengths so move further in the time the current is run.
  • The distance the DNA has migrated in the gel can be judged visually past monitoring the migration of the loading buffer dye.
  • The electric current is left on long plenty to ensure that the DNA fragments motility far enough across the gel to separate them, but not and so long that they run off the finish of the gel.

Analogy of Dna electrophoresis equipment used to separate DNA fragments past size. A gel sits within a tank of buffer. The Deoxyribonucleic acid samples are placed in wells at ane end of the gel and an electrical electric current passed beyond the gel. The negatively-charged DNA moves towards the postive electrode.
Image credit: Genome Research Limited

Visualising the results

  • Once the DNA has migrated far plenty beyond the gel, the electric current is switched off and the gel is removed from the electrophoresis tank.
  • To visualise the DNA, the gel is stained with a fluorescent dye that binds to the Deoxyribonucleic acid, and is placed on an ultraviolet transilluminator which will show upwardly the stained DNA as bright bands.
  • Alternatively the dye tin can exist mixed with the gel before it is poured.
  • If the gel has run correctly the banding pattern of the DNA marker/size standard will be visible.
  • It is and so possible to estimate the size of the DNA in your sample by imagining a horizontal line running beyond from the bands of the Dna marker. Yous tin can then guess the size of the Dna in the sample by matching them against the closest band in the marker.
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Illustration showing DNA bands separated on a gel. The length of the DNA fragments is compared to a marker containing fragments of known length. Image credit: Genome Research Limited

Analogy showing DNA bands separated on a gel. The length of the DNA fragments is compared to a mark containing fragments of known length.
Prototype credit: Genome Research Express

This page was last updated on 2021-07-21

Analyzing Dna by Gel Electrophoresis Allows Researchers to