Gel Electrophoresis gone Wrong

What is the depth of gel and how much buffer should be used when preparing an agarose gel? The recommended thickness for agarose gel is 3–4 mm; a gel thicker than 5mm will result in fuzzy bands and higher staining background. For any buffer the depth of buffer over the gel should range from 3 to 5 mm. Too much buffer will distort bands and cause heating and partial melting of the gel. Too little buffer and the gel is likely to partially dry out. Similarly, the amount of running buffer to cover over the gel in an electrophoresis apparatus is 3–5 mm. Too much buffer decreases DNA mobility and causes band distortion. Gel electrophoresis is a method for separation and analysis of macromolecules (DNA, RNA and proteins) and their fragments, based on their size and charge. It is used in clinical chemistry to separate proteins by charge or size (IEF agarose, essentially size independent) and in biochemistry and molecular biology to separate a mixed population of DNA and RNA fragments by length, to estimate the size of DNA and RNA fragments or to separate proteins by charge. Nucleic acid molecules are separated by applying an electric field to move the negatively charged molecules through a matrix of agarose or other substances. Shorter molecules move faster and migrate farther than longer ones because shorter molecules migrate more easily through the pores of the gel. This phenomenon is called sieving. Proteins are separated by the charge in agarose because the pores of the gel are too small to sieve proteins. Gel electrophoresis can also be used for the separation of nanoparticles. Gel electrophoresis uses a gel as an anticonvective medium or sieving medium during electrophoresis, the movement of a charged particle in an electrical current. Gels suppress the thermal convection caused by the application of the electric field, and can also act as a sieving medium, slowing the passage of molecules; gels can also simply serve to maintain the finished separation so that a post electrophoresis stain can be applied. 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. #NikolaysGeneticsLessons #gelElectrophoresis #gelElectrophoresisExplained #gelElectrophoresisOfDna #gelElectrophoresisProcedure #gelElectrophoresisLab #gelElectrophoresisAnalysis #agaroseGelElectrophoresis #agaroseGelElectrophoresisProtocol #agaroseGelElectrophoresisOfDna #agaroseGelElectrophoresisExplained #dnaGelElectrophoresis #dnaElectrophoresis #electrophoresisOfDna #sdsPageGelElectrophoresis #polyacrylamideGelElectrophoresis