BC2004 Lab Exercise 11 Spring Semester 2005
Gel Electrophoresis of DNA
weeks of 4/18-4/22 and 4/25-4/29/05
Gel electrophoresis is the most widely used method in molecular biology for separating macromolecules from one another on the basis of size. It is especially useful for analyzing mixtures of proteins or of nucleic acids with respect to the presence and relative abundance of molecules of different sizes, and for estimating the size of purified macromolecules; it can also be used as a step in purification of a specific protein or a specific length-class of DNA.
Electrophoresis gels for nucleic acids are most commonly prepared with agarose; gels for proteins are most commonly prepared with polyacrylamide. In this laboratory, agarose gel electrophoresis will be used to separate DNA molecules that differ in number of nucleotides and, therefore, in length (size). With a few changes in details (gel preparation, buffers used, stains applied, etc.), the same general approach would used be separate proteins on the basis of molecular weight on a polyacrylamide gel.
The fundamental principle of gel electrophoresis is that charged macromolecules will migrate through a gel when an electrical field is applied. Remember that DNA (in a neutral or basic environment) is negatively charged from the O- on the phosphate groups of the sugar-phosphate backbone. Because DNA is negatively charged, it will move toward a positive electrode and away from a negative electrode.
The distance migrated by a DNA molecule during the time it is subjected to the electrical field is determined by three factors: 1) the size (length) of the molecule; 2) the electrical field (dimensions and voltage differential); and 3) the density of the gel matrix. When a sample containing DNA molecules of different sizes is applied as a mixture to the same position in the gel, all of the molecules are subjected to the same voltage differential, and all of the molecules are challenged to work their way through the same gel matrix. This leaves molecular size as the only factor that will determine the final relative positions of the DNA molecules present in the original mixture.
In environments of pH greater than 7, DNA molecules carry an abundance of negative charges that are uniformly distributed along the length of each molecule. When subjected to an electrical field under neutral or basic conditions (with a pH > 7), DNA molecules migrate toward the positive pole (the anode) of the electrical field. In a gel matrix, the movement of DNA molecules in response to the electrical field is impeded by the gel, which acts like a sieve that slows the rate of their migration.
The larger the molecule, the more difficult it is for the molecule to work its way through the gel, so that smaller molecules move through the gel faster than larger molecules. As smaller molecules get ahead of larger molecules, they also get closer to the anode, and their migration accelerates in proportion to their proximity to the anode: the closer the molecule gets to the anode, the faster it migrates. Consequently, as smaller molecules of DNA get ahead of the larger molecules, the relative rate of migration of the smaller molecules is determined by two factors:
(a) their relative ease of working their way through the gel matrix, and (b) their increasing proximity to the positive pole of the electrical field. This compounding of influences on the rate of migration results in an exponential relationship between molecular size and distance migrated (see standard curve construction, below).
Gel electrophoresis results in the separation of DNA molecules of a mixture according to their molecular length (size), with the smaller molecules moving ahead of the larger molecules, which trail behind the smaller ones in order of increasing size. Wherever there are many DNA molecules of very nearly the same size, those molecules accumulate as a migrating “band” that is separated from smaller molecules ahead of them in the lane of their migration, and larger bands behind them.
The positions of the bands can be visualized by staining the gel with a dye that binds to DNA molecules, and the size of the molecules in each band can be estimated by comparison of the band’s position (distance migrated) with the positions of DNA molecules of known size. In this technique, a standard curve serves as the means of estimating the size of the molecules, in parallel to the use of standard curves for estimating the concentration of smaller molecules, as you learned in Exercises 1 and 2 of this laboratory course.
There is, however, one notable difference between the standard curves of Exercises 1 and 2 and the standard curve that you will construct in this exercise: the relationship between absorbance and solute concentration (in the useful range of the standard curves) was linear, whereas the relationship between molecular size and distance migrated in an electrophoretic gel is exponential, as explained above. For this reason, the standard curve that you construct in this exercise will be plotted using semilogarithmic coordinates so that the curve generated will relate the logarithm of molecular size to distance migrated plotted on a linear scale.
In this exercise, you will analyze DNA molecules in five preparations – two PCR reaction mixtures from Exercise 9 and three restriction endonuclease digests from Exercise 10. You will first mix your DNA samples with a dye called SYBR-Green, which binds to DNA and fluoresces when exposed to the proper wavelengths. You will then load each of your five DNA samples and a solution of DNA molecules of known sizes (your “Markers”) into wells in a gel prepared with 0.7 % agarose in 0.5 X TBE buffer, mounted in an electrophoresis apparatus and submerged in 0.5 X TBE buffer at pH 8. When all preparations (six for your group and six for another group of students) have been loaded into your gel, the electrophoresis apparatus will be closed, electrical leads will be attached, and sufficient current will be applied to maintain a voltage differential of approximately 120 V across the gel.
Electrophoresis will continue until the indicator dye approaches the positive end of the gel. This will probably require approximately 45 minutes. After the gel has been run, you will disconnect the electrical leads and expose the gel to the appropriate wavelengths of visible light. These wavelengths will cause the SYBR-Green (bound to the DNA) to fluoresce. You will mark the location of the fluorescent bands of DNA and estimate their length in base pairs.
Credits: The restriction digestion performed in Exercise 10 and analyzed here is based on a procedure developed by the DNA Learning Center of Cold Spring Harbor Laboratory that accompanies Carolina Biological Supply Company’s Restriction Mapping of Lambda DNA Kit (catalog no. 21-1173).
BC2004, Spring Semester 2005, Lab Exercise 11-1
Procedures
Do Parts B-D first. Your instructor will then demonstrate gel preparation. Following the demonstration, prepare one gel (Part A) for each group of 4 students (2 pairs). At some point during the lab period, your instructor will demonstrate how to transfer a gel to the visualization light box (you’ll need to be VERY careful because your gel is VERY fragile).
A. Preparation of an agarose gel.
Because your time in the lab is short, the gel you and your partners use will be ready for you at the beginning of your lab period. Nevertheless, in order for you to learn the steps in gel preparation, you and your partners will prepare one gel that will be used by the next lab section.
1. Close the ends of a gel tray with tape. The attachment must be watertight, so press the tape firmly against the ends of the tray. Insert the comb that forms the wells at one end of the tray.
2. Weigh 0.7 g agarose onto a piece of weighing paper (it works best to fold the paper in half and then open it up again so that you can easily pour the powder off the paper without spilling it; don’t forget to tare the balance after putting on the paper and before adding the agarose). Transfer your weighed agarose into a 250-ml Erlenmeyer flask. Measure 100 ml 0.5X TBE buffer in a graduated cylinder. Pour the buffer into the flask and mix by swirling gently.
3. Close the flask with a wadded up KimWipe or two (you’ll need to leave some room for air to come up so the flask doesn’t explode in the microwave) and heat it for 1 minute in the microwave oven.
4. Using a hot-glove to protect your hand, remove the flask and gently swirl the contents. Examine the contents to be certain that the agarose is completely melted; if it is not (and the solution looks even a little bit grainy), return the flask to the oven for additional heating. When the agarose has been completely melted, remove the flask to a water bath at 65o C to cool for a few minutes.
5. When the molten agarose has cooled to approximately 65o C, swirl it gently to be certain that the agarose is evenly distributed, and then gently pour the contents of the flask into the prepared tray. Rupture any air bubbles by poking them with the top of a clean pipet tip while the agarose is still molten.
6. Allow the gel to stand until it appears to be firm and is slightly cloudy, and then turn in the gel for use by the next lab section.
B. Preparation of the DNA solutions.
Your 2-PCR mixtures and 3-restriction digests have been stored in the freezer since your lab exercise 9 and 10 sessions. Retrieve them from your instructor and examine them to be certain that they have thawed completely.
1. Restriction digests. Using a fresh pipet tip for each addition, place 10 μl 3X SYBR-Green Loading Buffer into each digest tube. Mix the contents by gently aspirating the mixture into the pipet tip and then expelling the mixture back into the tube – once only. Avoid bubbles!
Lambda DNA is predominantly two-stranded, but when it has been cut, it will have one-stranded sticky ends. As a consequence, some of the molecules in your digests will reassociate with each other, making fragments unsuitable for accurate determination of restriction fragment length. In the absence of DNA ligase, as in our mixtures, these reassociated molecules are held together only by hydrogen bonds, which can be disrupted by heating the digests at 65oC for 10 min.
Therefore, heat your dye-digest mixtures in the 65oC incubator for 10 minutes immediately before loading them into your gel.
tube / amount of SYBR-Green Loading Buffer / amount and identity of restriction digest volumeDigest A / 10 uL / 20 uL ApaI
Digest E / 10 uL / 20 uL EcoO1091
Digest A/E / 10 uL / 20 uL ApaI/EcoO1091
BC2004, Spring Semester 2005, Lab Exercise 11-1
2. PCR mixtures and markers (do not heat these samples before loading them into the gel).
a. Obtain three 0.5-mL tubes. Label them “N,” “R,” and “M.”
b. Pipet 5 μl SYBR-Green 3X loading buffer into each tube (one pipet tip will do for this step because you are loading dye into clean tubes).
c. Using a fresh pipet tip for each addition, place 10 μl of DNA into each tube, according to its label (“M” is for the Markers, “N” is the PCR mixture missing the template DNA, and “R” is the PCR mixture complete with template DNA). Mix the contents by gently aspirating the mixture into the pipet tip and then expelling the mixture back into the tube. (Be gentle and avoid creating bubbles.)
tube / amount of SYBR-Green Loading Buffer / amount and identity of samplePCR N / 5 uL / PCR with no template DNA
PCR R / 5 uL / PCR complete with template DNA
M / 5 uL / markers
C. Loading the gel.
Load 15 μl of each of your five samples into the wells in your gel, in the following positions.
Load only 6 μl of the BenchTop 1kb DNA Markers/Ladder, “M,” on the chart below.
well number / 1 (or 9) / 2 (or 10) / 3 (or 11) / 4 (or 12) / 5 (or 13) / 6 (or 14)DNA sample / PCR “N” / PCR “R” / “M” / Digest A / Digest A/E / Digest E
(*Two pairs of students will load their preparations into one gel. One pair of students will use lanes 1 through 6, and the other pair of students will use lanes 9 through 14. Cross out the lane numbers in this table that you and your partner are not using.)
As soon as all twelve preparations have been loaded, notify the instructor. The current should be started as soon as possible after the wells are loaded; otherwise, the DNA molecules begin to diffuse into the gel in all directions, making the bands appear fuzzy (you also have to stay in lab until after the gel has finished running. The sooner you start your gel, the sooner it will be finished).
Should you attach the positive or negative lead to this end?
D. Electrophoresis