gel Electrophoresis

gel electrophoresis and its steps

1. Gel electrophoresis By: Jenifermatheew B.tech biotechnology

2. Gel electrophoresis: Electrophoresis simply means, the movement of charged molecule under the electric field Gel electrophoresis is the most common method used for the separation and purification of DNAs, RNAs, and protein. Principle: It works on the principle of when the electric current,is passing across a gel, it pulls small molecule through the gel. It is carried out in a electronic instrument called electrophoresis apparatus. Usually agarose gel is used to separate DNAs, RNAs and polyacrylamide gel is used in DNA sequencing and protein separation. The pore size of agarose gel is determined by concentration of agarose; low concentration of agarose gives large sized pores in the whereas high concentration gives small sized pores .

3. Gel electrophoresis involve the following steps : Agarose ( 0.6-1.5g/100ml) is dissolved in distilled water, boiled for 10 minutes and then cooled to 65`c The dye ethidium bromide(0.5ug/ml) is added to it and mixed well. The open ends of the casting tray are sealed with a tape. The teflon well forming comb is fitted at one end of the casting tray in such a way as to form wells at the top of the gel . The gel is then poured into the casting tray into the height of 4-6 mm. Kept as such for about 30- 60 minutes to solidify the gel The comb and sealing tape are removed carefully and the casting tray is then placed on the elevated platform of electrophoresis tank. Isolated DNA is cut with the restriction enzyme and mixed with bromophenol blue. This loaded into the well. Tris-borate EDTA buffer is poured into the reservoir so as the gel gets immersed to a height of 5 mm.

4. continue... The electrophoresis tank is closed with the glass shield and connected with the power apply . As the current passes from cathode to anode, it carries the carries the DNAs towards anode at the bottom of the gel. Electrophoresis blue reaches the bottom of the gel . The power is switched off and the gel is taken from the electrophoresis tank. The gel is visualized under a UV light illuminator . The DNA band stained with ethidium bromide show fluorescent bans .

5. Advantage : the gel is easily poured, does not denature the samples. The samples can also be recorded. Disadvantage : The disadvantages are that gels can melt during electrophoresis , the buffer can become exhausted, and different forms of genetic material may run in unpredictable forms.