Agarose Gel Electrophoresis
MATERIALS REQUIRED: Agarose, TBE Buffer, 6X Sample Loading Buffer. DNA ladder standard,Electrophoresis chamber, Power supply, Gel casting tray and combs, Glove,6X DNA Loading Dye - 10 ml. , Pipette and tips
PRINCIPLE:
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Gel electrophoresis is a widely used technique for the analysis of nucleic acids and proteins. Most every molecular biology research laboratory routinely uses agarose gel electrophoresis for the preparation and analysis of DNA. We will be using agarose gel electrophoresis to determine the presence and size of PCR products. Electrophoresis is a method of separating substances based on the rate of movement while under the influence of an electric field. Agarose is a polysaccharide purified from seaweed. An agarose gel is created by suspending dry agarose in a buffer solution, boiling until the solution becomes clear, and then pouring it into a casting tray and allowing it to cool. The result is a flexible gelatin-like slab. During electrophoresis, the gel is submersed in a chamber containing a buffer solution and a positive and negative electrode. The DNA to be analyzed is forced through the pores of the gel by the electrical current. Under an electrical field, DNA will move to the positive electrode (red) and away from the negative electrode (black). Several factors influence how fast the DNA moves, including; the strength of the electrical field, the concentration of agarose in the gel and most importantly, the size of the DNA molecules. Smaller DNA molecules move through the agarose faster than larger molecules. DNA itself is not visible within an agarose gel. The DNA will be visualized by the use of a dye that binds to DNA.PROCEDURE:
1. 6X DNA Loading Dye - 10 ml:
Reagents needed: 25mg bromophenol blue (0.25%), 25mg xylene cyanol (0.25%), 4g sucrose (40%), adjust volume to 10 ml with H2O.
Directions:
1) A volume of 25 mg of bromophenol blue was added to 6.7 ml of ddH2O and mix.
2) A volume of 25 mg of xylene cyanol FF was added and mixed.
3) A volume of 3.3 ml of glycerol was added and mixed.
4) Aliquot and was freezed at -20 °C for long-term storage.
2. Agarose Gel Electrophoresis Protocol:
Preparing the agarose gel:
• A volume of 0.24g Agarose powder was measured and was added to a 80ml flask
• A volume of 30ml TBE Buffer to the flask. (the total gel volume well vary depending on the size of the casting tray)
• The agarose was melted in a microwave or hot water bath until the solution becomes clear. (if using a microwave, heat the solution for several short intervals - do not let the solution boil for long periods as it may boil out of the flask).
• The solution was allowed to cool to about 50-55°C, swirling the flask occasionally to cool evenly. After that a volume of 1.5ul of micro liter EtBr was added.
• The ends of the casting tray was sealed with two layers of tape.
• The combs were placed in the gel casting tray.
• The melted agarose solution was poured into the casting tray and allowed to cool until it is solid (it should appear milky white).
• The combs were carefully pulled out and the tape was removed.
• The gel was placed in the electrophoresis chamber.
• Enough amount of TBE Buffer was added so that there is about 2-3 mm of buffer over the gel.
Loading the gel:
• A volume of 3 ul of 6X Sample Loading dye was added to each 20 ul DNA Sample.
• The order in which each sample will be loaded on the gel was recorded, including who prepared the sample, the DNA template - what organism the DNA came from, controls and ladder.
• Carefully a volume of 20 ul of each sample/Sample Loading Buffer mixture was pipetted into separate wells in the gel.
• A volume of 10 ul of the DNA ladder standard was pipetted into at least one well of each row on the gel.
(Note – if you are running multiple gels, avoid later confusion by loading the DNA ladder in different lanes on each gel.)
Running the gel:
• The lid was placed on the gel box, connecting the electrodes.
• The electrode wires were connected to the power supply, making sure the positive (red) and negative (black) are correctly connected. (Remember – “Run to Red”)
• The power was turned on , power supply to about 100 volts. Maximum allowed voltage will vary depending on the size of the electrophoresis chamber.
• It was checked to make sure the current is running through the buffer by looking for bubbles forming on each electrode.
• It was checked to make sure that the current is running in the correct direction by observing the movement of the blue loading dye – (note:this will take a couple of minutes (it will run in the same direction as the DNA)).
• The power was allowed to run until the blue dye approaches the end of the gel.
• The power was turned off.
• The wires were disconnected from the power supply.
• The lid of the electrophoresis chamber was removed.
• The tray containing gel was removed carefully using gloves & the gel was observed under UV Light -transilluminator.
RESULT: The gel was observed under UV Light -transilluminator.
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