Laboratory Exercise #5: Agarose Gel Electrophoresis

Introduction

Electrophoresis is a process which allows the separation of molecules (such as DNA) based on size. Electrophoresis means to “carry with electricity”. In DNA agarose gel electrophoresis, fragments of DNA are made to move using an electric field through a gel made of the polysaccharide agarose. The electric field runs through the gel by means of a conductive buffer solution and consists of a negative charge at one end of the gel (the anode) and a positive charge at the other end (the cathode). Because DNA is negatively charged, the DNA fragments are pushed away from the negative anode (due to repulsion) and attracted toward the cathode. The agarose functions as a molecular sieve, or a matrix of holes, through which smaller fragments of DNA can pass more easily (and quicker) than the larger fragments. Therefore, the rate at which a DNA fragment passes through the agarose gel is inversely proportional to its size.

Agarose is a polysaccharide (primarily of galactose) of an approximate molecular weight of 120,000 Daltons or 120KDa. When solidified, it forms a three-dimensional mesh of channels or pores of diameter ranging from 50 nm to >200 nm depending on the concentration of agarose used. Higher concentrations of agarose will yield lower average pore diameters. The 3-D structure is held together with hydrogen bonds and can therefore be disrupted by heating back to a liquid state.

The agarose gel is prepared in a buffer called TAE buffer (see Appendix for composition). This buffer is comprised of Tris-Cl, Acetic acid and EDTA that has been titrated to a pH of 8.0. A typical agarose gel of concentration between 1 to 2% will be capable of separating a wide range of DNA fragment sizes (50 to 20,000 base pairs). To prepare a 1% agarose gel, 1 g of agarose is added to 100 mL TAE buffer. However, the agarose will not dissolve in room temperature TAE but heating this mixture to near boiling will allow for the breaking of the agarose chain hydrogen bonds and the dissolution of the agarose particles in the TAE buffer. The dissolved agarose is then poured into a mold (often called a gel tray) and a “comb” is placed into the mold. As the agarose cools, it will polymerize inside the tray and around the comb. When the comb is pulled from the polymerized agarose gel, multiple wells are created for the addition of the DNA you wish to separate. These wells allows for the separation of multiple DNA samples, each of which running in parallel “lanes”.

To separate the DNA fragments in the prepared agarose gel, the gel (in the tray) is placed in an electrophoresis chamber called a gel box or tank. The gel tank is designed with a negative cathode at one end (black end) and a positive anode at the other (red end). When connected to a power supply, an electric field can be created between these two electrodes.

The electric field of an electrophoresis tank requires a conducting agent, which is the same TAE buffer used to make the gel. The tank is filled with TAE and the gel is placed in the tank. Before the gel can be “loaded” with the DNA fragments to be separated, the sample is mixed with a loading buffer. A typical DNA loading buffer contains 1% SDS to inactive any enzymatic reactions associated with the DNA sample, glycerol to make the DNA “heavy” so that it will sink to the bottom of the well and two indicator dyes, such as xylene cyanol (green color) and bromophenol blue (blue color).

DNA is colorless which makes it impossible to track its progress through an agarose gel. This might not be an issue if there was something to stop the DNA fragments from eventually “running off” the end of agarose gel and into the surrounding buffer. There isn’t. If run long enough, the DNA fragments can run out of the bottom of the gel!!! Therefore, the indicator dyes are an indirect way of tracking DNA progression. The two dyes will move through the agarose gel at different rates because, like the DNA fragments, they have different sizes. For example, xylene cyanol is bigger than bromophenol blue and it will not progress as far into the agarose gel as the bromophenol blue. In an agarose gel of 1%, bromophenol blue runs as if it is a DNA fragment with a size of 400 bp while xylene cyanol runs as if it is 3000 bp. As the gel runs, the distance between the two indicator dyes will increase. The further these two dyes are away from each other, the longer the gel has run and the better resolution of your DNA fragments you will achieve. One of the goals of a DNA gel is good resolution. Increased resolution means that you will be able to distinguish DNA fragments of similar sizes within the gel. Good resolution also requires prolonged durations of electrophoresis. So think of the indicator dyes as a built-in timer telling you when to remove your gel.

Once mixed with the loading dye, the prepared DNA is then loaded into the agarose gel wells and the gel is then connected to a power source. When an electric current is applied, the DNA fragments migrate toward the positive cathode. Because the agarose gel is porous, the DNA fragments must travel through these pores. The larger DNA molecules move more slowly through the gel while the smaller molecules move faster. When the gel is stopped after a sufficient amount of time (as assessed by the distance between the visible indicator dyes) and the DNA visualized, the different sized DNA fragments will form distinct bands on the gel.

Until recently, visualizing the DNA in an agarose gel required the addition of Ethidium Bromide (EtBr) to the heated agarose solution. EtBr works by intercalating between the bases of the DNA. When exposed to UV light, EtBr glows orange, allowing the visualization of the DNA fragments. However, EtBr is highly toxic and carcinogenic because it can structurally distort the DNA helix and stop replication and transcription. In fact similar intercalating agents are used as chemotherapy drugs! Therefore, EtBr-containing gels must be handled very carefully. Luckily today, there are many non-toxic DNA dyes that do the same thing as EtBr without endangering the user. One such dye, Fast Blast DNA dye will be used in today’s lab exercise.

The agarose gel is capable of separating DNA fragments into distinct bands. Each band represents a DNA fragment with a specific size. Bands in different lanes that end up the same distance from the top of the gel contain DNA fragments that passed through the gel with the same speed, which usually means they are approximately the same size. However, while these fragments are the same size, their actual size is still not known. To determine DNA fragment size, they are compared to molecular weight size markers called DNA ladders. The DNA ladder contains a mixture of fragments of known sizes. If such a ladder is run in one lane of the gel next to the unknown samples, the bands observed can be compared to those of the unknown in order to determine their size.

Pre-lab activities

Record the purpose of this activity.

READ THE PROTOCOL. Be sure you understand what each step in this protocol is for.

TAE buffer preparation

Most TAE buffers are prepared as a concentrated stock. In the lab, you will find a bottle of TAE labelled as 50XTAE. This is the 50XTAE that you prepared in the beginning of the course. The 50X indicates that it is 50-times more concentrated than what you would use. In order for you to prepare your gels and run them, you must dilute the concentrated TAE to a “working concentration”. Working concentrations are 1X concentrations and are what are actually used in a lab protocol.

This means that you must dilute your 50X TAE, fifty times to achieve a 1X TAE working solution. You will probably require 50 mL of 1X TAE for gel preparation and at least 400 mL of 1X TAE to run the gel. Therefore, you should prepare at least 500 mL of 1X TAE. Any “leftovers” can be stored in a large plastic reagent bottle labelled 1X TAE found at the electrophoresis station. This 1X TAE will be used in future electrophoresis protocols so BE CAREFUL HOW YOU MAKE THIS. Your future experiments will depend on your accuracy!!

You will prepare your 1X TAE using the 50X TAE stock and by diluting it with water.

  1. In your notebook, calculate how much 50X TAE you will need to make 500 mL 1X TAE.
  2. Measure out this volume of 50X TAE using a graduated cylinder and pour it into a glass beaker
  3. Determine the remaining volume of water that you will need.
  4. Measure this out with a graduated cylinder and add it to your 50X TAE.
  5. Set this 1X TAE aside.

Lab Materials

Common workstation

Micropipettes

Pipet tips

Agarose powder

50X TAE

10X DNA loading dye

250 mL Erlenmeyer flasks

Heat resistant gloves

Glass beakers

Graduated cylinders

Large glass bottles

Laboratory tape

Permanent markers

Microwave

Thermometer

Weighing workstation

Electronic balances & weigh boats

Electrophoresis workstation

Agarose powder

50X TAE

Agarose gel electrophoresis set-up

a. Gel box

b. Gel tray

c. Gel comb

Laboratory tape

Power supplies

Millimeter rulers

Large reagent bottles for leftover 1X TAE

Heat resistant gloves

Protocol

  1. 1% Agarose gel preparation
  1. From the common station, obtain a horizontal gel tank, lid, agarose gel tray, comb and power supply and bring it to your lab bench.
  2. Prepare the gel tray as shown by your professor. Make a note in your lab notebook as to how you prepared the gel.
  3. Insert the comb into the tray.
  4. To prepare 50 mL of 1% agarose, weigh out 0.5 g of agarose using a weigh boat and carefully pour the agarose powder into a clean, empty 250 mL Erlenmeyer flask.
  5. Measure out 50 mL of 1XTAE and carefully pour it into the flask.
  6. Carefully swirl the mixture.

NOTE: You will notice that the agarose will not dissolve in room temperature. You will need to heat the agarose to near boiling.

  1. Place the flask into the microwave and heat for 1 minute. Open the microwave and with a heat resistant glove, carefully swirl to mix the solution. Do this slowly and carefully!!!
  2. Continue to heat and swirl in 1 minute increments until the agarose is completely dissolved in the TAE.

CAUTION: As the agarose solution reaches near boiling, rapid swirling of the flask will cause the solution to “bubble over” out of the flask. Second degree burns may result. Therefore, swirl slowly and gently as the agarose begins to reach near boiling. NEVER handle the flask without a heat resistant glove or suitable hand protection.

  1. Measure and record the temperature of the “melted” agarose solution in your lab notebook.
  2. Let the agarose solution cool for 5 minutes. Measure the temperature again and record it.
  3. When the gel has cooled to at least 65°C, carefully pour the cooled agarose into the prepared gel tray with the inserted comb. Ensure the tray is on a level surface and that you do not disturb the gel as it is polymerizing.

NOTE: If you were using Ethidium Bromide to stain your DNA fragments, you would add it at this step. However, you will be using a non-toxic DNA stain.

  1. Let polymerize.

NOTE: As the agarose polymerizes, it will transform from a clear solution to an opaque gel. Polymerized gels may be wrapped in Saran wrap and placed at 4°C until needed.

  1. Loading the gel into the electrophoresis tank
  1. Once polymerized, carefully pull out the comb to create the wells and remove the tape around the gel tray.
  2. Place the tray in the electrophoresis tank so that the exposed ends of the gel face the electrodes and so that the wells are closest to the black electrode.

Laboratory Technique: Place the electrophoresis tank so that the black electrode faces up and away from you. To remind yourself, remember the phrase “black top”.

  1. Slowly fill the tank with the 1XTAE running buffer. Fill the tank so that the entire surface of the gel is covered with 1XTAE. A “layer” of TAE on top of the gel with a depth of 2mm is sufficient.

Laboratory Technique: As the 1XTAE covers the surface of the gel, the wells will fill with TAE. This is known as “flooding” the wells. The flooded wells are ready to be loaded.

  1. Preparing your samples & running the gel
  1. Obtain a tube of 10X DNA loading dye, a tube of DNA ladder, a P20 micropipette and pipette tips and bring them to your bench. Also obtain your PCR reactions that you ran in the last protocol.
  2. If you have never loaded a gel before, your professor will have a few agarose gels set-up for you to practice with. Using these gels, pipette 10ul of gel loading dye into the first well on the left side of the gel. Your professor will demonstrate the proper technique for pipetting a sample into your well.
  3. Practice loading a couple of wells.
  4. Let your partner practice loading a couple of wells also.
  5. Once you are comfortable with loading a gel, load your gel with your prepared PCR reactions.
  1. Prepare your PCR samples by transferring 10ul of the PCR reaction into a clean and properly labelled1.5mL centrifuge tube and adding 2.0uL of DNA loading dye. Be sure to label your centrifuge tubes properly so you don’t mix-up your PCR reactions when you run them on the gel
  2. Vortex your samples to mix with the loading dye and “flick” spin using the microcentrifuge to collect the prepared samples at the bottom of the tube.
  3. Load the gel as indicated below. Lane #1 will be the well on the left as you look at the gel.

Lane 1 = 5.0uL of DNA ladder

Lane 2 = 10.0uL of PCR reaction #A

Lane 3 = 10.0uL of PCR reaction #B

Lane 4 = 10.0uL of PCR reaction #C (reaction A made with an RTU bead)

Lane 5 = 10.0uL of PCR reaction #D (reaction B made with an RTU bead)

Lane 6 = 10.0uL of PCR reaction #N (no-template negative control)

9. Once you have finished loading, place the electrophoresis lid on the tank and connect it to the power supply.

NOTE: The electrophoresis tank will have a built-in black and a red electrode. The lid will also have a black and a red electrode. Make sure the colors match when you are attaching the lid. Also make sure to match up the electrode color and the colored receptacle in the power supply.

10. Turn the power supply setting to high and turn it on.

11. You will run this gel for approximately 30 minutes. At 5 minute increments, turn off the power, remove the lid and measure the distance between the xylene cyanol dye front and the bromophenol blue dye front. Make note of these measurements in your lab notebook. Re-connect the power supply and keep running the gel. Compare your results to other lab teams.

12. When complete, turn off the power supply, disconnect the leads and remove the lid from the tank.

13. Wearing gloves, carefully remove the gel in its tray.

For a video of running a DNA agarose gel – check this out this video from Bio-Rad

D. Staining the gel with the InstaStain Ethidium Bromide cards

1. Wearing gloves, remove and discard the clear plastic protective sheet from the unprinted side of the InstaStain card. Place the exposed, unprinted side of the card on the gel. Be sure the majority of the surface of the gel is covered with the card.

CAUTION: Ethidium bromide (EtBr) is a carcinogen. NEVER handle EtBr-containing materials without wearing PPE. ALWAYS dispose of EtBr-containing materials in the appropriate waste container. Your professor will provide this container.

2. With a gloved hand, remove any air bubbles between the card and the gel by gently running your fingers over the entire surface of the gel. Alternatively, you may gently roll a glass Pasteur pipette up and down the surface of the gel

3. Stain the gels for 3 to 5 minutes – but NO MORE than 5 minutes.

4. Remove the InstaStain card. Dispose of the card in the indicated waste container.

5. Examine the gel using a long UV-wavelength trans-illuminator (300nm). The DNA bands will appear as bright orange bands.

CAUTION: UV irradiation can damage skin and eyes. NEVER visualize DNA bands on a trans-illuminator without the protective eye shield in place. Do not place unprotected skin on the UV surface.

6. Take a picture of your gel, print it out and paste it into your lab notebook. Label it.

Interpretation of Protocol – Lab Notebook

In your lab notebook, document the protocol you have just performed and include any important observations such as the compositions of any reagents used, the appearance of you gel. Use the questions below to help you document this protocol. You may also choose to take pictures at key points in the performance of this protocol and paste these pictures into your lab notebook.

  1. How long did it take for the agarose to dissolve in the heated 1XTAE?
  2. What temperature did the 1XTAE have to reach before the agarose dissolved completely?
  3. What did the dissolved agarose solution look like versus the polymerized agarose gel?
  4. Make any relevant technique notes regarding your loading of the gel.
  5. What do you think might happen if you accidently put the lid on backwards so that the tank’s black electrode becomes connected to the red electrode on the lid? What do you think might happen if you hooked up the gel to the power supply and did not match up the colors?
  6. Which of the two dye fronts ran the fastest? What can you conclude about the sizes of the two dyes used in your loading gel?

7. Using the picture of your gel, identify those bands from PCR reactions. Estimate their sizes and note it in the table you created in your lab notebook.