Principle Gel Electrophoresis is a method used in the laboratory for the separation of charged molecules like nucleic acids (DNA and RNA) and proteins that differ in size or charge (Agarose gel electrophoresis (basic method)). LB (Lithium Borate) buffer is the widely used buffer in agarose gel electrophoresis. The buffer solution comprises of Lithium hydroxide monohydrate and boric acid. LB buffer has low conductivity which can run at a high speed (5-50V/cm) than the gels which are made of TBE or TAE buffers (5-10 V/cm) which in turn reduces the run time drastically (Brody 6).
Experimental Required
Materials: Gel Electrophoresis Unit (Gel casting unit and Combs); Weighing balance; Conical flask; Microwave and Pipettes (0.5µl - 10µl; 20µl).
Consumables: Agarose powder; Lithium hydroxide monohydrate; Ethidium bromide; 1Kb plus DNA ladder; Gel loading buffer and Filter tips (10µl; 20µl).
Procedure
Preparation of 10L of 20X LB Buffer: Measure 9.0L of distilled water and add 83.92g of Lithium hydroxide monohydrate and 360g of Boric acid. Adjust the pH to 8.2 and make up the volume to 10L.
Assembling the gel electrophoresis unit: Place the horizontal gel apparatus on a level surface. Open the lid of the gel apparatus and place the buffer tray on the tray support stand. Arrange the casting tray in the center of the buffer tray in such a way that the outermost well visualization strip is towards the negative electrode. Place the gel casting dams in the grooves of the buffer tray and apply pressure on either side to seal the casting tray. Insert appropriate combs into the slots provided.
Preparation of the 1% agarose gel: Take 5ml of the 20X LB buffer and add 95ml of dH20 to make 100ml of 1X LB running buffer. Add into a 250ml conical flask, 1g of agarose powder and pour 100ml of 1X LB running buffer, swirl slowly. Place the conical flask in the microwave and heat for 2 min until the agarose melts completely. Allow the gel solution to cool and add 5µl of ethidium bromide dye and mix well. Pour the mixture into the gel casting tray and allow it to solidify.
Running the Gel: Fill the tank with 1X LB running buffer so that the gel is completely submerged and carefully remove the combs. Load 5µl of 1Kb plus DNA ladder into one of the well. Mix 20µl samples with 2µl of gel loading dye which would help the sample to sink into the wells. Load the samples into individual wells and place the lid on the gel apparatus. Switch on the power pack and set 300 voltage. Run the samples from anode to cathode for 7 min. When the marker dye has reached an appropriate distance, turn off mains supply and disconnect the power leads. Place the gel on a UV transilluminator to visualize and analyze the DNA bands.
Works Cited
Lewis, Matt. “Agarose gel electrophoresis (basic method).” Web. 2001.
Brody, Jonathan R., and Scott E. Kern. “History and principles of conductive media for standard
DNA electrophoresis.” Analytical Biochemistry 333.1 (2004): 1-13.
