Preparation of agarose gel for DNA analysis

Overview

  • Agarose gel electrophoresis is a very common method in all molecular biology laboratories. It has many applications including analysis of DNA (size analysis, detection of DNA in a sample, separation and purification of DNA fragments etc.).
  • Agarose gel is a gelatin like slab, which contains small wells. It is prepared by melting agarose in a suitable electrophoresis buffer.
  • Percentage of agarose in the gel decides the pore size through which DNA moves. Electrophoresis buffer facilitates the passage of electric current through gel.
  • Melted agarose is poured in a specialized tray (casting tray), which control the size of the gel (the amount of agarose solution is dependent primarily on size of the casting tray). Comb is used to create wells in agarose. gel. Well facilitates the agarose gel to keep the DNA sample.
  • Solidified agarose gel is submerged into electrophoresis buffer. Since DNA is negatively charged and moved towards the positive electrode, side of the gel with wells is always placed towards the negative electrode. A electrophoresis power supply apparatus is used to maintain electric field in the electrophoresis tank.
  • Movement of DNA is primarily controlled by size and form (conformation) of DNA, agarose concentration in the gel and strength of electric field. Often a small amount of ethidium bromide is added in the agarose gel to visualize DNA. Alternatively gel can be placed in a ethidium bromide containing solution to visualize DNA after electrophoresis.

Requirements

Reagents:

  • Agarose
  • Ethidium bromide solution (10 mg/ml in water)
  • Electrophoresis buffer (TAE or TBE Buffer)

Equipment and disposables:

  • Gel casting tray and combs
  • Micropipettes and tips
  • Gloves

    Objective

Preparation a 0.8 % agarose gel (Gel size – 8 cm X 10 cm X 0.5 cm) in TAE buffer

Note : Depending on the need, one can prepare various percentage of agarose gel (See table). Here we have taken an example to describe the preparation process clearly.


Preparation:

Step 1: Dissolve the agarose in electrophoresis buffer

  • Weigh out 0.4 gm agarose in a conical flask. Add 50 ml 1X TAE buffer in this. Suspend the agarose by swirling the flask. Wait for 1 – 2 min to allow hydration of agarose particles.
  • Melt the agarose in microwave or hot plate until the solution becomes clear.

Tips

  1. To make 0.8 % agarose gel of size 8 cm (width) X 10 cm (length) X 0.5 cm (thickness), 50 ml solution is sufficient.
  2. The total gel volume varies depending on the size of the casting tray. To decide how much volume of agarose solution is required for a casting tray, one can use water to get an idea about the volume of agarose solution required.

Precautions:

  1. The size of the flask should be atleast 5 times of the volume of the agarose solution to be prepared. While heating, solution boils and comes outside. So if the size of the flask is not appropriate, one may lose some part of the solution.
  2. If microwave or hot plate is used to melt the agarose, heat the solution for several short intervals. Avoid continuous boiling of the solution as it may boil out of the flask. Alternatively one can use boiling water bath to melt the agarose. It may take longer time.
  3. Make sure that melted agarose solution appear clear and transparent, devoid of any suspended particles of agarose. If there are some suspended particles, melt it more.
  4. Sometimes there is a significant loss of water, which depends on the melting procedure. Measure the total volume of melted agarose solution, and if the volume is significantly less, make up the loss by adding deionized water (Do not add buffer). The best way is to place the flask to weighing balance just before starting the melting process and set the balance zero. After melting agarose, again place the flask containing melting agarose solution on the balance. You will see negative reading, which will be equal to the loss of water during the process of melting agarose. Adjust this negative value to 0 by adding deionized water to the melted agarose solution.

Step 2: Add Ethidium bromide to agarose solution (optional)

  • Cool the solution until the temperature reaches 50-55°C. Swirl the flask occasionally to cool solution evenly. One can prepare the casting tray during this time.
  • Add 2.5 μl ethidium bromide in the solution. Mix by gentle swirling. Avoid air bubble formation.

Precautions:

  1. Ethidium bromide is carcinogenic. Use appropriate safety measures (use latex gloves, wear lab coat) to avoid any harm.
  2. Do not add Ethidium bromide when the solution is very hot.

Step 3: Pour the melted agarose solution into a casting tray

  • Prepare the casting tray by sealing both the open ends of tray with tape. Pour the melted agarose solution into a casting tray. Insert the comb at appropriate place.
  • Wait until agarose is solidified completely. Solidified agarose gel will appear milky white.

Tips:

  1. One can place the comb before pouring the agarose solution into the casting tray. 2: Agarose gel can be stored for several days. To store the agarose gel, we recommend not to remove comb and tape. Dip the agarose gel in the TBE buffer so that it contains moisture. Seal the gel in a plastic wrap. Store in the cold room (4°C). In case gel becomes dry, allow it to rehydrate with in the electrophoresis (TAE or TBE) buffer for few minutes. Before starting electrophoresis, let it come to room temperature.
  1. Precautions:
    1. Avoid air bubble. Remove air bubble with the help of pipette tip.
    2. While inserting the comb, take care that teeth of comb should not touch the bottom of casting tray. If it touches, Well will be like a hole. In this case, sample will come out from the well.