Electrophoresis Animation

Gel electrophoresis is a technique used for the separation of DNA, RNA, or protein molecules using electric current applied to gel matrix.It is used has as a preparative technique prior to use of other methods such as mass spectrometry, RFLP, PCR, cloning, DNA sequencing, or Southern blotting


Gel electrophoresis is performed in silica gel which inert porus medium. In electrophoresis macromolecules like DNA, RNA and protein migrate when electric current is passed through the medium. Separation of molecules depends upon two forces namely mass and charge, When the macromolecules are mixed with a buffer and applied to a gel, the electric current from one eletrode refuses the molecule while the other one attracts the molecule, this frictional force separates the molecule by size, During the process of electrophoresis molecules are forced to move through the pores when a electrical current is applied, the molecules speed depends on the strength of the field, their shape, size and strength and temperature of the buffer,separeted molecules can be seen in bands



After the electrophoresis is complete, the molecules in the gel can be stained to make them visible. Ethidium bromide, silver, or coomassie blue dye may be used for this process. Other methods may also be used to visualize the separation of the mixture's components on the gel. If the analyte molecules fluoresce under ultraviolet light, a photograph can be taken of the gel under ultraviolet lighting conditions. If the molecules to be separated contain radioactivity added for visibility, an autoradiogram can be recorded of the gel.

If several mixtures have initially been injected next to each other, they will run parallel in individual lanes. Depending on the number of different molecules, each lane shows separation of the components from the original mixture as one or more distinct bands, one band per component. Incomplete separation of the components can lead to overlapping bands, or to indistinguishable smears representing multiple unresolved components.


Bands in different lanes that end up at the same distance from the top contain molecules that passed through the gel with the same speed, which usually means they are approximately the same size. There are molecular weight size markers available that contain a mixture of molecules of known sizes. If such a marker was run on one lane in the gel parallel to the unknown samples, the bands observed can be compared to those of the unknown in order to determine their size. The distance a band travels is approximately inversely proportional to the logarithm of the size of the molecule.













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What is Southern Blot

Southern blot is a method routinely used in molecular biology to check for the presence of a DNA sequence in a DNA sample. Southern blotting combines agarose gel electrophoresis for size separation of DNA with methods to transfer the size-separated DNA to a filter membrane for probe hybridization. The method is named after its inventor, the British biologist Edwin Southern.The southern blot is used to verify the presence or absence of a specific nucleotide sequence in the DNA from different sources and to identify the size of the restriction fragment that contains the sequence.
In this procedure, the DNA is isolated from each source and then digested with a specific restriction enzyme. The DNA restriction fragments are then loaded onto an agrose gel and the fragments separated by electrophoresis according to size, with the smaller fragments migrating faster than larger fragments. The DNA is then transferred from the fragile gel to a nylon filter.






Next the radioactively labeled nucleic acid probe is added. The probe binds to complementary DNA segments. Note that the DNA segment being probed is not present in organism B

To detect the position of the radioactive probe, the nylon membrane is covered with an X-ray film. After development, the positions of the probe become visible.

Northern blot

Northern blot is a technique used in molecular biology research to study gene expression. It takes its name from its similarity to the Southern blot technique, named for biologist Edwin Southern. The major difference is that RNA, rather than DNA, is analyzed in the northern blot. Both techniques use electrophoresis and detection with a hybridization probe. The northern blot technique was developed in 1977 by James Alwine, David Kemp, and George Stark at Stanford University.

A northern blot is very similar to a Southern blot except that it is RNA rather than DNA which is extracted, run on a gel and transferred to a filter membrane. There are 3 types of RNA: tRNA (transfer RNA - active in assembly of polypeptide chains), rRNA (ribosomal RNA - part of the structure of ribosomes) and mRNA (messenger RNA - the product of DNA transcription and used for translation of a gene into a protein). It is mRNA which is isolated and hybridized in northern blots.








* mRNA is extracted from the cells grown in galactose and cells grown in glucodse and purified.
* The mRNA is loaded onto a gel for electrophoresis. Lane 1 has gal mRNa Lane 2 has the Glucose mRNA.
* An electric current is passed through the gel and the RNA moves away from the negative electrode. The distance moved depends on the size of the RNA fragment. Since genes are different sizes the size of the mRNAs varies also. This results in a smear on a gel. Standards are used to quantitate the size. The RNA can be visualized by staining first with a fluorescent dye and then lighting with UV.
* RNA is single-stranded, so it can be transferred out of the gel and onto a membrane without any further treatment. The transfer can be done electrically or by capillary action with a high salt solution.
* A GAL DNA probe is incubated with the blot.the single stranded GAL DNA probe binds with immobilized GAL mRNA The blot is washed to remove non-specifically bount probe and then a development step allows visualization of the probe that is bound.