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Electrophoresis is a technique commonly used in the lab to separate charged molecules, like DNA, according to size.
- Gel electrophoresis is a technique commonly used in laboratories to separate charged molecules like DNA?, RNA?and proteins? according to their size.
- Charged molecules move through a gel when an electric current is passed across it.
- An electric current is applied across the gel so that one end of the gel has a positive charge and the other end has a negative charge.
- The movement of charged molecules is called migration. Molecules migrate towards the opposite charge. A molecule with a negative charge will therefore be pulled towards the positive end (opposites attract!).
- The gel consists of a permeable matrix, a bit like a sieve, through which molecules can travel when an electric current is passed across it.
Smaller molecules migrate through the gel more quickly and therefore travel further than larger fragments that migrate more slowly and therefore will travel a shorter distance.
Gel electrophoresis and DNA
- Electrophoresis enables you to distinguish DNA fragments of different 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 being arranged in order of size.
- The use of dyes, fluorescent?tags or radioactive? labels enables the DNA on the gel to be seen after they have been separated. They will appear as bands on the gel.
- A DNA 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 DNA marker, you can work out the approximate length of the DNA fragments in the samples.
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 are working with.
- The higher the agarose concentration, the denser the matrix and vice versa. Smaller fragments of DNA are separated on higher concentrations of agarose whilst larger molecules require a lower concentration of agarose.
- To make 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 then poured into a gel casting tray and a “comb” is placed at one end to make wells for the sample to be pipetted into.
- Once the gel has cooled and solidified (it will now be opaque rather than clear) the comb is removed.
- Many people now use pre-made gels.
- The gel is 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 electric 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 will prevent it from floating out of the wells and so that the migration of the sample through the gel can be seen.
- A DNA marker (also known as a size standard or a DNA ladder) is loaded into the first well of the gel. The fragments in the marker are of a known length so can be used to help approximate 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 across the gel to the positive end).
Separating the fragments
- The electrical current is then turned on so that the negatively charged DNA 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 by monitoring the migration of the loading buffer dye.
- The electrical current is left on long enough to ensure that the DNA fragments move far enough across the gel to separate them, but not so long that they run off the end of the gel.