Gel electrophoresis separates DNA fragments according to their length.Method
1. An agarose gel is made in a tank, with small wells cut at one end. The gel is covered with a buffer solution that conducts electricity.
2. The DNA samples are mixed with a loading dye and placed in the wells using a micropipette. A DNA ladder (a mixture of fragments of known lengths) is placed in one well for comparison.
3. An electric current is passed through the gel. The wells are at the negative electrode (cathode) end.
4. DNA is negatively charged because of the phosphate groups in its sugar-phosphate backbone, so the fragments move through the gel towards the positive electrode (anode).
5. The gel acts like a mesh. Smaller fragments move through it more easily, so they travel further in a given time than larger fragments.
6. The current is switched off before the fastest fragments reach the end of the gel. Fragments of the same length finish in the same place, forming a band.
DNA is colourless, so the bands must be made visible. The gel can be stained with a dye that binds to DNA and fluoresces under ultraviolet light, or the DNA can be labelled with fluorescent tags during PCR. Alternatively the DNA is transferred to a membrane and located using labelled probes.Estimating fragment size
The distance each band has travelled is compared with the bands of the DNA ladder. A band that has travelled the same distance as the 500 base pair band of the ladder is about 500 base pairs long.Gel electrophoresis can also be used to separate proteins and RNA.
1. An agarose gel is made in a tank, with small wells cut at one end. The gel is covered with a buffer solution that conducts electricity.
2. The DNA samples are mixed with a loading dye and placed in the wells using a micropipette. A DNA ladder (a mixture of fragments of known lengths) is placed in one well for comparison.
3. An electric current is passed through the gel. The wells are at the negative electrode (cathode) end.
4. DNA is negatively charged because of the phosphate groups in its sugar-phosphate backbone, so the fragments move through the gel towards the positive electrode (anode).
5. The gel acts like a mesh. Smaller fragments move through it more easily, so they travel further in a given time than larger fragments.
6. The current is switched off before the fastest fragments reach the end of the gel. Fragments of the same length finish in the same place, forming a band.
DNA is colourless, so the bands must be made visible. The gel can be stained with a dye that binds to DNA and fluoresces under ultraviolet light, or the DNA can be labelled with fluorescent tags during PCR. Alternatively the DNA is transferred to a membrane and located using labelled probes.Estimating fragment size
The distance each band has travelled is compared with the bands of the DNA ladder. A band that has travelled the same distance as the 500 base pair band of the ladder is about 500 base pairs long.Gel electrophoresis can also be used to separate proteins and RNA.