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.
  • Agarose concentration in a gel is expressed in percentage of agarose to the volume of buffer (w/v). For example, 1% agarose gel contains 1 gram agarose in 100 ml of electrophoresis buffer. Most commonly used gel is in the range of 0.5–2%.
  • Percentage of agarose decides the pore size of the gel through which DNA moves. As the concentration of agarose in a gel increases, pore size decreases.
  • Electrophoresis buffer facilitates the passage of electric current through the gel. Most common electrophoresis buffers are TBE and TAE.
  • Agarose is insoluble in cold water/electrophoresis buffers. In order to dissolve agarose, agarose suspension in electrophoresis buffer is heated to melt agarose particles. When the melted solution is allowed to cool to room temperature, it forms a gel. The whole process is called gelation.
  • Melted agarose is poured in a specialized tray (casting tray), which control the size of the gel. The comb is used to creating wells in agarose gel. Wells facilitate the agarose gel to keep the DNA sample.
  • Solidified agarose gel is submerged in 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. An electrophoresis power supply apparatus (Power Pack) is used to maintain the 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 ethidium bromide containing the solution to visualize DNA after electrophoresis.

Requirements

  • Reagents
    •  Agarose
    • Ethidium bromide solution (10 mg/ml in water)
    • Electrophoresis buffer (TAE or TBE Buffer)
    • Deionized/distilled water
  • Equipment and disposables
    • Gel casting tray and combs
    • Micropipettes and tips
    • Gloves
    • Measuring cylinder

Note: Prepare the casting tray by sealing both the open ends of the tray with tape. Pour water in the tray to check whether the tray is sealed properly and is not leaky.

Related content:

Objective

Preparation a 0.8 % agarose gel (gel size – 10 cm X 12 cm X 0.4 cm) in TAE buffer or TBE buffer
Notes:
  • Depending on the need, one can prepare various percentage of agarose gel (0.5 – 2%). Here we have taken an example to describe the preparation process clearly.
  • Use low percentage of agarose gel to resolve high molecular weight DNA and high percentage to resolve low molecular weight DNA.
  • Use 0.5X TBE buffer or 1X TAE buffer for running and preparation of agarose gel. Always use the same running buffer in which the agarose gel is prepared.

Procedure:

Step 1: Weigh out 0.4 gm agarose in a conical flask/bottle. Add 50 ml of 1X TAE buffer. Suspend the agarose by swirling the flask. Wait for 1 – 2 min to allow hydration of agarose particles.
Notes:
  • To make 0.8 % agarose gel of size 10 cm (width) X 12 cm (length) X 0.4 cm (thickness), 50 ml solution is required.
  • The volume of the flask/bottle should be 3 – 4 times the volume of the agarose solution being prepared.
Tip:
  • The total gel volume varies depending on the size of the casting tray. Use the following formula to calculate the volume of agarose solution:

Total volume of agarose solution = width of casting tray x length of casting tray x thickness of gel.

Step 2: Weigh the flask/bottle.

Note:
  • Sometimes, there is a significant loss of water during the melting process, which depends on the melting procedure. You can calculate the loss of water by weighing the flask/bottle just before and after the melting process. Loss of water is significantly high when you prepare agarose suspension in conical flask/beaker (no lid) and melt it in a microwave to melt agarose suspension.
Step 3: Melt the agarose in microwave or hot plate until the solution becomes clear.
  • While heating, swirl the flask occasionally.
Tip:
  • Heat the solution for several short intervals instead of boiling continuously. Continuous boiling can cause the solution to boil out of the flask.
Precaution:
  • Make sure that melted agarose solution appear clear and transparent, devoid of any suspended particles of agarose. Melt it more If there are some suspended particles.
Step 4: Weigh the flask/bottle again and make up the loss by adding deionized/distilled water (do not add buffer).
Step 5: Cool the solution until the temperature reaches 55 – 60°C.
  • Swirl the flask occasionally to cool solution evenly.
Tip:
  • You can store the solution in a 60°C water bath to prevent overcooling of agarose. This can take 15 – 20 min. Casting trany can be prepared during this time.
Step 6 (optional): Add Ethidium bromide to agarose solution
  • Add 2.5 μl ethidium bromide in the solution. Mix by gentle swirling. Avoid air bubble formation.
Precautions:
  • Ethidium bromide is carcinogenic. Use appropriate safety measures (wear latex gloves and lab coat) to avoid any harm.
  • Do not add Ethidium bromide when the solution is very hot.
Step 7: Pour the melted agarose solution into the casting tray
  • Pour the melted agarose solution into the casting tray.
  • Insert the comb at appropriate place.
  • Wait until agarose is solidified completely. Solidified agarose gel will appear milky white.
Tips
  • One can place the comb before pouring the agarose solution into the casting tray.
  • Remove air bubbles with the help of pipette tip.
Precautions:
  • 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.

Agarose gel is ready for use.

  • Agarose gel can be stored for few 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 and store in the cold room (4°C). Before starting electrophoresis, let it come to room temperature.
Agarose amount required for the preparation of agarose solution of specific concentration and volume
Volume (ml)/Conc (%)
0.5%
0.8%
1.0%
1.2%
1.5%
2.0%
30 ml
0.15 gm
0.24 gm
0.3 gm
0.36 gm
0.45 gm
0.6 gm
40 ml
0.2 gm
0.32 gm
0.4 gm
0.48 gm
0.6 gm
0.8 gm
50 ml 0.25 gm 0.4 gm 0.5 gm 0.6 gm 0.75 gm 1.0 gm
100 ml
0.5 gm
0.8 gm
1.0 gm
1.2 gm
1.5 gm
2.0 gm
200 ml 1.0 gm 1.6 gm 2.0 gm 2.4 gm 3.0 gm 4.0 gm
500 ml 2.5 gm 4.0 gm 5.0 gm 6.0 gm 7.5 gm 10.0 gm