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DNA gel electrophoresis is usually performed for analytical purposes, often after amplification of DNA via polymerase chain reaction (PCR), but may be used as a preparative technique prior to use of other methods such as mass spectrometry, RFLP, PCR, cloning, DNA sequencing, or southern blotting for further characterization.
An agarose gel cast in tray, to be used for gel electrophoresis. Agarose gel is a three-dimensional matrix formed of helical agarose molecules in supercoiled bundles that are aggregated into three-dimensional structures with channels and pores through which biomolecules can pass. [3]
PCR (polymerase chain reaction) is a DNA amplification technique that can be applied to various types of fragments. Prior to this development, to amplify DNA, it had to be cloned or isolated. Shortly after the discovery of PCR came the idea of using PCR-based markers for gel electrophoresis.
For fluorescent dyes, after electrophoresis the gel is illuminated with an ultraviolet lamp (usually by placing it on a light box, while using protective gear to limit exposure to ultraviolet radiation). The illuminator apparatus mostly also contains imaging apparatus that takes an image of the gel, after illumination with UV radiation.
After restriction digest, DNA can then be analysed using agarose gel electrophoresis. In gel electrophoresis, a sample of DNA is first "loaded" onto a slab of agarose gel (literally pipetted into small wells at one end of the slab). The gel is then subjected to an electric field, which draws the negatively charged DNA across it.
After further PCR cycles, to amplify the DNA, the sample can be separated by agarose gel electrophoresis, followed by electroelution for collection. Efficiently generating oligonucleotides beyond ~110 nucleotides in length is very difficult, so to insert a mutation further into a sequence than a 110 nt primer will allow, it is necessary to ...