Category Archives: Electrophoresis

Running DNA samples in an agarose gel

Overview:

  • Agarose gel electrophoresis is a very common method of analyzing DNA in most molecular biology laboratories.
  • DNA sample is mixed with DNA loading dye/buffer and placed in wells of agarose gel. Loading buffer helps DNA sample to settle at the bottom of the wells.
  • Since DNA is negatively charged due to its phosphate group, it moves towards positive pole under the 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.
  • Movement of DNA is primarily controlled by size and form (conformation) of DNA, agarose concentration in the gel and strength of electric field.
  • Higher percentage (2%) agarose gel is used to resolve small size DNA fragments whereas lower percentage agarose gel is used to resolve large DNA fragments.
  • DNA fragments lower than 50 bp are separated by polyacrylamide gel electrophoresis. Larger DNA fragments (more than 30 kB) are separated by pulse field gel electrophoresis.
Related content:

Requirements:

Reagents:
Equipment and disposables:
  • Micropipette and tips
  • Gloves
  • Electrophoresis Power supply
  • Electrophoresis apparatus
  • UV transilluminator or Gel doc

Note: Use the same electrophoresis buffer which is used to prepare agarose gel (e.g., Use TAE if the agarose gel is prepared in TAE).

Protocol:

Step 1: Place the agarose gel with casting tray in the electrophoresis tank. Since DNA moves from negative to positive electrode, the side where the wells are, should be towards the negative electrode.

Note:
  • Different kinds of gel casting apparatus are available. In some cases, you need to seal the edges with the tape, whereas, in others, you need to just place casting tray into a cassette.
Precautions:
  • If casting tray is sealed with tape, remove the tape from both sides. Tape will not allow the passage of electric current through gel.
  • Don’t remove the gel from the casting tray. This may cause damage to the wells.

Step 2: Fill the electrophoresis tank with electrophoresis buffer.

Tips:
  • We recommend you to add ethidium bromide to a final concentration of 0.2 – 0.5 μg/ml in the electrophoresis buffer if the the agarose gel contains ethidium bromide. For example, add 20 – 50 µl ethidium bromide (stock conc 10 mg/ml)  in 1000 ml electrophoresis buffer.
  • As the electrophoresis progress, positively charged ethidium bromide moves towards negative electrode, resulting in depletion of ethidium bromide from the rear end of the agarose gel. This results in the appearance of dark zone in the agarose gel when you analyze gel under uv trans-illuminator.
  • Use the same electrophoresis buffer for running gel which was used to prepare agarose gel (e.g., Use TAE if the agarose gel was prepared in TAE)
  • If you are using TAE electrophoresis buffer, pour 1X TAE buffer in the tank. For TBE, use 0.5X TBE electrophoresis buffer.
Precautions:
  • Agarose gel should be submerged in buffer, but don’t fill electrophoresis buffer too much. Too much electrophoresis buffer over the agarose gel can cause slow run and distorted DNA band.
  • Ethidium bromide is carcinogenic. Use appropriate safety measures (wear latex gloves and lab coat) to avoid any harm.

Step 3: Carefully remove the comb from the gel without causing damage to the wells.

Note:
  • The comb can be removed even before placing the gel in the electrophoresis buffer, but occasionally, this can cause collapsing of wells. It is much safer to remove the comb after submerging the gel in electrophoresis buffer. Electrophoresis buffer moves inside the wells as the comb is removed, thus protecting the wells from collapsing.
Precaution:
  • Wells should not be damaged. Often wells are damaged at the bottom that may go uncheck, causing the leakage and loss of samples. If the samples are precious, check the well by loading a small amount of 1X loading dye in the wells.

Step 4: Prepare the DNA sample by mixing 5 volume of DNA solution with 1 volume of 6X DNA loading dye (e.g., 5 μl DNA solution and 1 μl DNA loading dye). Mix it and load into the wells of agarose gel carefully without spilling the sample into the adjacent wells.

Tips:
  • If DNA sample need to be diluted, one can use water or more conveniently the electrophoresis buffer from the tank.
  • It is very convenient and economical to mix the DNA solution with loading dye on the parafilm instead of using microcentrifuge tubes. To mix the sample with dye, one can place the required amount of dye on the parafilm. Now add DNA solution and mix it by few rounds (4 – 5 times) of pipetting.
Precautions:
  • Do not destroy well while sample loading.
  • While pipetting DNA sample, avoid air-bubbles. Air-bubbles can cause spillage of DNA sample into adjacent wells.

Step 5: Connect the electrophoretic apparatus to power supply and start the gel run.

Notes:
  • Place the lid on the gel box, and connect electrodes with power supply using electric wires, supplied with the electrophoresis apparatus. Set the current maximum and voltage 70 – 100 volt. Turn the power supply on.
  • The distance between electrodes in electrophoretic apparatus determines the maximum voltage. Voltage should not exceed 5 volts/cm. Since most commonly used electrophoresis devices have distance 14 – 20 cm between electrodes, 70 – 100 volt is default voltage.
Tip:
  • By looking air-bubbles from the electrodes (negative electrode), one can ensure that the electric supply is functional.
Precautions:
  • Always place the lid of electrophoretic apparatus to avoid electric shock.
  • Make sure that connections [to positive (red in colour) and negative poles (blue in colour)] to power pack is proper. If it is reverse, sample will run on the opposite direction and will come out from the gel. The direction of the run can be monitored by observing the movement of the loading dye. (it will run in the same direction as the DNA).

Step 6: Run the agarose until the purple dye (represents Bromophenol blue) approaches the end of the gel or 3/4 of the gel. Turn off the power supply and disconnect the wires from the power pack.

Note:
  • Depending on the DNA size and resolution of DNA fragments, one has to decide the gel run time.

Step 7: Once the gel run over, turn off the power supply and disconnect the wires. Take out the gel from the electrophoresis chamber and analyze under UV transilluminator or Gel documentation system.

Tip:
  • Immediately analyze the gel for DNA just after you disconnect electric supply. Storing gel in electrophoresis buffer can cause loss of resolution due to diffusion of DNA band.
Precautions:
  • Use hand gloves and follow safety rules as the gel contains ethidium bromide.
  • Use tray to take out gel. Some casting trays allow direct visualization of gel as they are permeable to UV light but are costlier. If casting tray is impermeable to UV light, take out the gel and analyze under UV light.
  • UV is harmful. Use UV Safety Goggles and shield to protect yourself from direct exposure to UV rays.

 

 

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