How long does the DNA separation take



    Gel electrophoresis is an analytical method used in chemistry and molecular biology to separate different types of molecules. A mixture of molecules to be separated migrates under the influence of an electric field (see:Electrophoresis) through a gel which is in an ionic buffer solution. Depending on the size and charge of the molecules, they move at different speeds through the gel, which acts as a molecular sieve. Small, negatively charged molecules (anions) migrate fastest in the direction of the positively charged anode and positively charged molecules (cations) in the direction of the negatively charged cathode.

Gel matrix

The molecules of the gel, for example agarose (see agarose gel electrophoresis) or polymerized acrylamide (polyacrylamide; see polyacrylamide gel electrophoresis), form a close-meshed network that prevents the molecules to be separated from migrating in the electric field.

Agarose gels have relatively large pores (150 nm for 1%, 500 nm for 0.16% gels) and are well suited for the separation of DNA and high molecular weight proteins. Polyacrylamide gels have much smaller pores (3-6 nm). The pore size depends on the acrylamide concentration and the degree of crosslinking.

Depending on the application, various additives are added to the gel, for example SDS for SDS polyacrylamide gel electrophoresis (SDS-PAGE) or ampholytes for isoelectric focusing (IEF). However, it is also not uncommon to do without the SDS, as SDS is added to the samples in the sample buffer before they are applied.

Gels can actually be made yourself without much effort, although it requires some experience to get the gel tight right away. Finished gels and the corresponding buffer systems can also be purchased commercially.

execution

Classic gel electrophoresis is carried out as zone electrophoresis. One method of achieving higher resolution is batch electrophoresis.

Gel electrophoresis generates heat. This must be removed to ensure optimal conditions. Therefore, the gel electrophoresis should be carried out in cooled apparatus at constant temperatures in order to achieve reproducible results.

Ideally, the electrophoresis is ended when the smallest or most mobile molecules have reached the end of the gel. This guarantees the highest possible separation of the molecules.

evaluation

To evaluate the gel after electrophoresis, the molecules to be separated are either radioactively labeled before electrophoresis and then detected in an autoradiography or after electrophoresis "colored" with various dyes such as ethidium bromide and viewed under UV light. This is what you do with DNA fragments, for example. Proteins can be stained (see: Fairbanks staining, silver staining) and / or can be detected immunologically (Western blot).

The same molecules run in discrete zones - colloquially as Gangs designated - by the gel. Several samples can run parallel to each other through the same gel at the same time. If the size of some molecules is known, one can estimate the size of the other molecules by comparing their bands with the remaining bands. Such molecular weight standards are commercially available.

A determination of the amount of a substance in a band or the relative proportion of a band (please refer: Quantification) is possible after staining the gel and a subsequent densitometric evaluation. Evaluation software is used in most cases to determine the measured values ​​of a gel, such as widths, MW weights, quantifications or normalization.

Areas of application

Gel electrophoresis is widely used in molecular biology and biochemistry:

1see also:Southern blot, Northern blot
2see also:Western blot

Category: electrophoresis