Principle
Separation of negatively charged nucleic acid molecules based on their charged by mass ratio on agarose gel in the presence of an electric field.
Instrumentation
Power pack: A power pack which supplies a constant DC current and a voltage of 1.5 volts is selected. The power pack is turn connected to a stabilizer as even a minute fluctuation may decrease the resolution power of electrophoresis.
Electrodes: Platinum electrodes are used.
Buffer system: Discontinuous or continuous buffer tank can be used to separate nucleic acids.
Preparation
Agarose gels are prepared by boiling agarose in buffer and the gels are cast on a specially made tray called gel plat form. This gel plate form consists of plastic plates and glass plates. The open sides of the plate are covered by adhesive tape or a plastic frame. The plastic frame will be removed before placing. Before the gel is set a template or a comb is placed on the gel to form wells. The comb is coated with teflon, anodized aluminum, and Perspex. The gel is allowed to set for about 20-30 minutes at 37-38oc. once the gel is set the comb is removed. Removal of comb leads to formation of wells and the wells are the space where the sample is spotted. The gel is ready and it is placed in electrophoretic apparatus on elevated gel bed.
Sample preparation and loading
The sample solution is usually prepared in a suitable buffer. The sample solution also contains glycerol, sucrose or ficol to increase the density of the sample so that it sinks into the well. Also a marker dye like Bromophenol is added and electrophoresis is carried out. Sample is loaded into the wells using a micro syringe or micropipette.
Running or sample
Once the sample is loaded power pack is switched on and a constant voltage of 1.5 volts is supplied. Separation is based upon the length of the nucleic acid fragments. Electrophoresis is carried out overnight. Increase in voltage causes heating of gel and therefore a constant voltage is maintained.