Laboratory Exercise: ELECTROPHORESIS & MIGRATION DISTANCE

In order to analyze the products of digestion by restriction enzymes, it is necessary to visualize the fragments produced.  Gel electrophoresis is a technology that has been developed to separate DNA fragments, taking advantage of the inherent chemistry of the DNA molecule.  DNA is negatively charged due to the phosphate groups embedded in side rails of the helix.  When DNA is placed in an electric field, the fragments will migrate toward the positive pole (anode) as they are being repelled from the negative pole (cathode).

The sorting of fragment size takes place in an agarose gel.  Agarose is the starchy ingredient in agar, and is derived from an algae.  When agarose cools, it gels (think Jell-o) as the individual molecules interact.  These interactions form a 3-dimensional lattice structure with small pores randomly placed throughout the gel.  It is through these pores that the DNA molecules will migrate.  Smaller molecules will migrate faster, as there is less friction to the molecule as it moves toward the positive pole.  Larger molecules will lag behind as they will not pass through the pores as readily.  A row of holes, or sample wells, is placed along one side of the gel, and it is into thee that the DNA sample will be loaded.

The agarose gel is placed in an electrophoresis chamber and covered with a buffer solution containing ions that help pass the current through the gel.  Just prior to loading the DNHA sample, the digested DNA is mixed with a solution that consists of sucrose and one or more visible dyes.  The dense sucrose solution adds weight to the DNA sample, and helps it sink to the bottom of the well.  The dye does not interact with the DNA molecule, but migrates independently.  Due to its small size, it runs ahead of the DNA sample, providing a visible marker as to the progress of the DNA fragments.

Following electrophoresis, the gel is soaked in a stain that will diffuse throughout the gel and become concentrated in areas where it is binding to DNA fragments.  The excess stain is then soaked out of the gel, and the fragments will become visible.

In this lab you will:

·         Learn to pour a gel

·         Place DNA samples of lambda cut with HindIII into a gel

·         Analyze the gel to calculate DNA fragment size

Pouring the Gel:

1. Set up the gel box by placing a dam in each of the slots at the end of the gel bed.  Be sure that the side of the dam next to the gel box forms a right angle with the gel bed.

2. Seal the ends of the gel bed before pouring the agarose.  Use the plastic pipette to lay down a narrow bead of melted agarose along the joint between the black dam and the gel bed along both ends.

 

3. Wait one minute while the bead solidifies, then pour enough agarose into the gel bed until the level of agarose reaches the top of the bed but does not overflow (about 20mL).  The gel will appear thicker than it actually is – observe the level carefully and be sure to pour in enough agarose.
4. Immediately put an 8-tooth comb into the end of the gel that is nearest the black (negative) electrode.  The red stripe under the comb will allow you to visualize the wells after the comb has been removed.
5. Allow the gel to solidify at room temperature for 20-30 minutes.  Either:
   1. Proceed with loading the gels, or
   2. Remove dams and place gel in plastic bag with a small amount of buffer. Refrigerate overnight.  Do not remove the comb,

Loading the Gel:

1. Slowly remove dams.  Pour enough TAE buffer into the gel chamber to completely cover the gel.  Wait a few minutes before removing the comb.  The buffer will soften the gel slightly, allowing the comb to be removed without breaking the wells.  Pull the comb straight up.
2. Add 2µl of loading dye to the tube containing the DNA digest. 
   Flick vigorously or briefly pulse centrifuge to mix the contents.  The loading dye is already in the tube containing the DNA ladder. 
3. After loading 20µl of the DNA ladder in lane 1, load 20µl of each sample into the wells.

Running the Gel:

1.   Once the samples have been loaded, do not move the gel box and keep agitation to the gel to a minimum.  When all samples have been loaded, close the lid on the gel box and attach the electrical leads – red to red and black to black.  Be sure the power switch is off and the white dot on the rheostat knob is at the “min” position.  Turn the power on and adjust the voltage to approximately 110V using the rheostat.

2. Electrophorese the sample for 30n minutes, or until the leading edge of the dye has migrated to a point midway between the second and third red stripes. 
3. Turn down the rheostat, turn off the power supply, and disconnect the electrical leads from the gel box.

Staining the Gel:

1.   Wearing gloves, lift the gel chamber out of the electrophoresis box and pour excess buffer into a sink or beaker.  Pour approximately 100 ml Methylene Blue stain into a staining tray.  Carefully transfer the gel from the gel bed to the tray very slowly sliding the gel from the bed into the staining tray.  Leave the gel in the stain for approximately 30 minutes.

2. Transfer the gel into a tray that contains 100 ml distilled water.  Let sit a few minutes, then pour off water into a sink or beaker, and put fresh water in the tray to continue destaining – 30 minutes or overnight at 4C.
3. Drain excess water from the gel tray, and use a spatula to transfer the gel to the surface of the light box.  Align the wells with the zero point on the ruler that is taped to the surface of the light box.

Photographing the Gel:

1.   Take a Polaroid photo of your gel.  Check that the camera is set with an ‘f’ stop of 32 and a shutter speed of 15.  Fit the hood of the camera over the gel, turn on the illuminator light and squeeze the shutter of the camera.  Turn off the illuminator light and pull the film from the camera by first pulling the protruding white tab to move the film out of the cassette.  Next, grasp the black tab at the end of the film and slowly pull it out of the cassette.  Wait 30 seconds and peel the picture from the backing.

Analyzing the Gel:

The migration of DNA molecules in agarose gels is roughly proportional to the inverse of the log of their molecular weight (corresponding to the size of the fragments).  Using semi-logarithmic graph paper, calculate the fragment sizes as compared to the DNA ladder standard.

(insert one kilobase ladder standard information here)

1.      Plot the DNA ladder standard on the semi-logarithmic graph paper.  Plot the 4 size of each fragment (Y axis) against the distanse migrated in mm (X axis).  Draw a curve or line to connect the points plotted.

2.  Use the standard curve to estimate the sizes of the fragments produced by the lambda/HindIII digest.  Based on the number of fragments produced, how many HIndIII restriction sites are on the lambda plasmid?

TEACHER NOTES:

DESIGNED FOR: Groups of 4 students

TIME REQUIRED: (2-3) 40 minute class periods

MATERIAL LIST:

Preparation of Solutions:

Loading the gel:

This lab is designed to calculate the fragment size of lambda digested with HindIII.  You can also use the products of the genomic DNA lab digested with Eco R1 and/or the products of the pRAS2/pUC19 lab digested with EcoR1