4. INTRODUCTION Agarose gel electrophoresis isa method used in biochemistry and molecular biology to separate DNA; or RNA molecules by size. However, proteins can also be separated on agarose gels, Proteins are separated by charge in agarose because te pores ofthe ge are too larg to sieve proteins. Separation fhe molecules is achieved by moving negatively charged nucleic acid molecules through an agarose natrix in an electric field. The purpose of the gel might be to look at the DNA, to quantify itor fo rola a particular band. The DNA is visualised in the gel by the addition of ethidium bromide, This binds strongly to DNA by intercalating between the bases and is fluorescent meaning that it absorbs invisible UV light and transmits the energy as visible orange light. Figure 8.1: Set up for Agarose Gel ElectrophoresisBroouemennr Lanoearony Maine DNA samples 15k Figure 82: A eattern of ONA Bands Under UV Light 2. PROCEDURES 2a. Preparation of Agarose Gel i. Weigh out 0.5g of agarose into a 250 mL conical flask, ii, Add 50 mL. of 0.5xTBE, swirl to mix. iii, Carefully bring the solution just to the boil to dissolve the agarose. iv. Leave it to cool on the bench for 5 minutes down to about 60°C. vy. Add 5 ulethidium bromide stock (10 mg/ml) per 100 ml gel solution. The reason for allowing, the agarose to cool a little before this step is to minimise production of ethidium bromide ‘vapour. Ethidium Bromide is mutagenic and should be handled with extreme caution. Dispose of the contaminated tip into a dedicated ethidium bromide waste container, vi. Pour the gel slowly into the tank and push any bubbles away to the side using a disposable tip. vii. Insert the comb and double check that it is correctly positioned. viii. When the gel has cooled down and become solid, carefully remove the comb. The holes that remain in the gel are the wells or slots. Put the gel, together with the rack, into a tank with TAB. The gel must be completely covered with TAE, with the slots at the end electrode that will have the negative current. 46Expennenr 8/ Agarose Get ELecrRoPHORESS 2b. Electrophoresis i. Use a micropipette to inject about 2.5 pl of stained DNA (a DNA ladder is also highly recommended). ii, Close the lid of the electrophoresis chamber and apply current (typically 100 V for 30 minutes ‘with 15 ml of gel). fii, Check that a currents lowing and monitor the progress of the gel by reference to the marker dye. iv. Stop the gel when the bromophenol blue has run 3/4 the length of the gel. vy, _ Switch off and unplug the gel tank. 2c. Visualization of DNA bands i. Camy the gel (in its holder if possible) to the dark-room to look at on the UY light-box. Some sel holders are not UV transparent so you have to carefully place the gel onto the glass surface of the light-box. UV is eareinogenie and must not be allowed to shine on naked skin or eyes. ‘So wear face protection, gloves and long sleeves. RESULTS rem RESULT | Poriod of time (minutes) required for solidification of agarose gel | ‘Number of wells or slots that has been successfully | formed or fe rime) ae fr conpetn of | electrophoretic separation Number of DNA bands present in agarose gel Presence of smear in DNA profile a7