XVI.  ELECTROPHORESIS:  PREPARING AND RUNNING AGAROSE GELS

INTRODUCTION

          In this module you will separate DNA molecules according to their size, using agarose gel that you have prepared yourself.  However, before starting, please read the background material that follows.

  Gel electrophoresis

     Electrophoresis is a method that allows separation of molecules in an electric field on the basis of size/molecular weight and shape.  A molecule will migrate in an electric field if it has a net positive or negative charge.  A molecule with negative charge (anion) will migrate and be attracted to the positive electrode (anode), and a molecule with positive charge (cation) will migrate and be attracted to the negative electrode (cathode).

     Migration and separation of molecules are carried out using pre-cast “gels” composed of a solid matrix of molecules that contains microscopic pores.  Molecules are separated according to size and shape by migrating through the pores in the gel.  Separation is based on the different mobility through the gel of molecules with different size and/or shape.  The gel matrix retards movement of molecules by a seiving effect.  Small or compact molecules travel faster through the gel matrix than large or asymmetric molecules, which encounter more frictional resistance in the gel meshwork.

     Gel electrophoresis is a widely used technique in cell and molecular biology for separating biological macromolecules such as nucleic acids (DNA, RNA) and proteins.  The gel matrix for separating proteins is commonly polyacrylamide, a water-soluble cross-linked polymer.  Therefore, separation of proteins is usually done by polyacrylamide gel electrophoresis (PAGE).  Agarose, a complex linear polysaccharide, is routinely used as a matrix component for separating DNA and RNA.  Therefore, this gel system is known as agarose gel electrophoresis.  Separation of protein on polyacrylamide gels and nucleic acids on agarose gels generates banding patterns which can be visualized using various detection methods.

     The size of nucleic acid and protein molecules is typically measured in different units.  For DNA and RNA, the length is conveniently measured in “base pairs” (bps) or kilobase pairs (kbps).  For proteins the measurement is usually molecular weight in Daltons (Da) or kiloDaltons (kDa).  The sizes of DNA, RNA, or protein molecules separated on gels can be estimated by comparison with standard molecular weight markers of known size that were run in parallel with the unknown samples during gel electrophoresis.

          The relative mobility of molecules, i.e., how fast they travel through the gel relative to each other, depends on gel concentration as well.  Therefore, gels of differing concentrations, such as 1% and 2% agarose or 12% and 15% polyacrylamide, are used for separating different sized molecules.  The particular percent agarose or polyacrylamide needed depends on the size of the molecules that require separation.  Generally, the smaller the size, the higher the percentage of agarose or polyacrylamide needed.  At a given gel concentration, the distance traveled by a molecule will be inversely proportional to the log10 of the molecular weight or number of base pairs.  Thus, the larger or longer the molecule, the more time it takes to migrate through gel matrices, as noted earlier.  DNA and RNA molecules carry an intrinsic negative charge conferred by their phosphate backbones.  Proteins, however, by virtue of the different charges on each amino acid residue, will have different charges along their surface.  Therefore, for proteins, the mobility is proportional to molecular weight only if a detergent such as sodium dodecyl sulfate (SDS) is used to coat the protein surface and make charges uniformly negative along their length.

Agarose Gel Electrophoresis

     As noted above, DNA is a polyanion, carrying an innate negative charge conferred by its negatively charged phosphates along the DNA backbone.  Thus, in agarose gel electrophoresis, DNAS will migrate through agarose from the negative cathode towards the positive anode.

     Different size pieces of DNA will separate according to both their size and shape.  Lower molecular weight (lower length) DNAs will move faster through gel matrix pores than larger ones.  However, the shape of a DNA molecule also plays a role in its movement, with the fastest moving form known as “supercoiled” DNA.  It has the highest mobility because of the compactness of the superhelical shape.  Linear and circular DNA molecules will travel slower because they tend to interact to a greater extent with the gel matrix.  Thus, linear, circular, and supercoiled forms of DNA having the same number of base pairs will migrate at different rates through agarose gels.

     The resolution of DNA molecules by their size/shape using agarose gel electrophoresis is truly remarkable.  Good separation can be achieved with DNA molecules whose size differs by as little as 1%, and it is also possible to separate two DNA molecules that differ by only a single superhelical twist. Furthermore, the technique can be used with DNA molecules containing fewer than 10 bps to as many as 300,000 bps. 

     Gels of different agarose concentration must be used for different size ranges:

0.8 - 1.5 % agarose for DNA sizes up to 50,000 bps

0.2 - 0.4% agarose for very large DNA sizes

     While polyacrylamide gel electrophoresis is typically performed in a vertical apparatus, agarose gel electrophoresis is conducted in a horizontal configuration to provide better support at low agarose concentrations.  This produces less distortion (collapse) of the gel and of the DNA bands during electrophoresis.  The submarine system, in which the gel is completely submerged in buffer, is the easiest to operate.

     During electrophoresis water is electrolyzed, generating protons (H+) at the anode, and hydroxyl ions (OH-) at the cathode.  The cathode end of the electrophoresis chamber then becomes basic, and the anode end becomes acidic.  Use of a buffer system is therefore needed.  The two most popular buffers for agarose gels are Tris-Borate-EDTA (TBE) and tris-Acetate-EDTA (TAE).  Besides their excellent buffering capacity, they also aid in DNA mobility through the gel matrix.

     In this module, you will use the TBE buffer and a 1% agarose gel, running a mixture of dye markers as the sample.  These markers are test standards having properties resembling nucleic acids.  The “ChromatrackTM” dye markers are pipetted into one of the wells formed in the agarose gel, then allowed to electrophorese.  The unique dye marker mix contains 6 dye markers that will migrate at different molecular weights, providing a range of colored bands visible in ordinary room light.

     The following reference chart indicates the approximate bps with which the individual tracking dyes migrate at different agarose gel concentrations.

ChromatrackTM Migration Chart (in bps)

Dye        0.75%     1.0%      4.0%

blue      9,000      4,300      300

yellow     2,600     1,800      100

red       1,300        900       50

blue        950       600       25

pink        600        300       15

orange      200        100       <10

EXERCISE #1:  AGAROSE GEL ELECTROPHORESIS VIDEO

          View the video presentation on Agarose gel Electrophoresis

EXERCISE #2:  LOCATING MATERIALS

          Locate and check that you have all of the following materials to perform agarose gel electrophoresis:

On reagent shelf:

  i) Chroma TrackTM dye marker

  ii) agarose powder

In refrigerator:

  i) 10X TBE buffer stock solution

  ii) stirrer/heating unit

  iii) power supply

  iv) balance

   v) P20 pipetman and pipet tips

   vi) electrophoresis apparatus

     (gel tank and lid)

In gel electrophoresis drawer:

   i) gel casting tray with foam pads

  ii) gel running tray (detachable from casting tray)

  iii) comb to form wells

CAUTION: HIGH VOLTAGE IS USED IN ELECTROPHORESIS  

EXERCISE #3:  CASTING AND RUNNING AN AGAROSE GEL

Procedure:

1.  Weigh out agarose needed to make 30ml of a 1% gel.  (Reminder: % in this case is weight per unit volume, i.e, grams/100 ml)

2.  Place the weighed-out agarose in a 150ml beaker.

3.  Make up 280ml of 1X TBE buffer from stock 10X TBE.  The stock bottle is in the refrigerator.  Store buffer in a plastic storage bottle.

4.  Add 30ml of the 1X TBE to agarose in the beaker

5.  Add a stirring bar and place on stirrer/heating unit.  Set stirring to no. 2 and heating to no. 3.

6.  Stir gently to a slight boil, when agarose dissolves and the liquid is clear.

7.  Carefully remove flask with paper towel or hot glove around neck of flask, and place on bench.  Remove stirring bar with magnet remover.

8.  Allow gel to cool slightly. (All plastic parts of the electrophoresis unit are heat resistant to 65oC, but liquids should be cooled to about 50oC before pouring in order to prolong unit life).  Use a thermometer.

9.  Make sure that the gel running tray is safely tucked into the gel casting tray, with tray edges gently pressed against foam pads.

10. Pour agarose into gel running tray

11.  Add the comb to make wells, and allow the gel to solidify

12. Check that the power switch for the electrophoresis unit is OFF.

13. When gel hardens, carefully remove comb to expose wells

14. Remove the gel running tray from the casting tray and place it in the electrophoresis unit gel tank

15. Add the rest of your 1X TBE so that it just completely covers the gel.  Do not use excessive buffer, as it will short-circuit the system and lower the mobility

16. Using P20 pipetman, pipet 2µl of “ChromatrackTM” dye marker into a middle well.  Insert the pipet tip into the bottom of the well and expel liquid slowly.  Take care not to poke or puncture the agarose.

17. Connect the current electrodes (attached to the electrophoresis unit lid) so that the dye markers will migrate toward the positive anode, i.e., connect negative electrode at the position of the wells, and the positive electrode at the other end of the gel.

18. Turn ON power supply and set to 100V.  Band migration of dye markers can be observed as electrophoresis proceeds.

19. The gel should be run for 1 hour, until all the colored bands are well separated from each other.  To se the bands, turn OFF power supply, remove lid, and place a piece of white paper under the gel tank to see the colored bands clearly.

20. Referring to the migration chart, identify the corresponding colored bands and their approximate number of base pairs.

21. Remove the gel running tray (with gel in it) from the gel tank

22. Using a ruler, measure the distance traveled from the well (in cm) by each colored band.

23. Using semi-log paper, plot the number of base pairs of each colored band versus the distance traveled.  plot base pairs on the log scale, and cm migrated on the linear scale.  Draw the best line through these points to generate a standard curve.