Gel Electrophoresis
How a gel sorts DNA by size, choosing the agarose percentage and buffer, loading, staining and imaging, reading bands against a ladder, and troubleshooting.
Chapters
- 0:14 Where gel electrophoresis came from
- 1:32 How DNA moves and how the gel sorts it
- 3:13 Choose agarose or polyacrylamide and the percentage
- 4:54 Running buffers, TAE and TBE
- 5:43 Cast a gel
- 6:48 Loading dye and tracking dyes
- 8:01 DNA ladders
- 8:39 Load the gel, set the voltage and run time
- 9:40 Stain the gel
- 11:17 Image the gel
- 12:01 Read a band size against the ladder
- 13:50 Check a PCR product
- 14:52 Plasmid forms on a gel
- 15:58 How to approach troubleshooting
- 16:39 Smeared bands
- 17:51 No bands or faint bands
- 18:56 Smiling bands and damaged wells
- 19:55 Bands off the gel and extra bands
- 21:09 Safety
- 22:07 Summary and practice question
Transcript
0:14 Where gel electrophoresis came from
Electrophoresis is the movement of charged particles in an electric field, and its first major use was on proteins. Arne Tiselius wrote his doctoral thesis in 1930 on the moving boundary method for studying the electrophoresis of proteins. He received the 1948 Nobel Prize in Chemistry for discoveries about the complex nature of serum proteins. In a free solution with no support, the moving boundary method showed the advancing boundaries of a protein mixture and did not separate the proteins into distinct zones. A solid gel holds the sample in place while the field acts on it, which is why the technique moved from boundaries in solution to bands in a gel. The same problem applied to DNA, because DNA was sized mostly by sucrose density gradient centrifugation before agarose gels. Sucrose gradients gave only an approximation of size. In the early 1970s, laboratories applied agarose gels to DNA, and by 1973 agarose gels run with ethidium bromide were analyzing the fragments that restriction enzymes produce.
1:32 How DNA moves and how the gel sorts it
Agarose gels separate DNA fragments by size because of two properties, the charge of DNA and the structure of the gel. The phosphate backbone of DNA carries a negative charge, so in an electric field DNA moves toward the positive electrode, which is called the anode. Each nucleotide carries one phosphate and about the same mass. The ratio of charge to mass is therefore the same for a 100 base pair fragment and a 10 kilobase fragment, where a kilobase is one thousand base pairs. In free solution, the two would move at the same speed, and you could not tell the fragments apart. In a gel, the agarose matrix slows long fragments more than short ones. Agarose forms a mesh of pores. A small fragment passes through the pores with few collisions and travels far. A large fragment collides with the mesh more often and travels a shorter distance. The mesh resists large molecules more strongly than small molecules. The leading model for this movement is biased reptation. In biased reptation, the leading edge of the molecule moves forward and pulls the rest of the chain along. The distance traveled falls in step with the log of the fragment size. Plot log size against distance and the ladder bands fall on a straight line over the useful range of the gel. Several other factors also set the rate at which DNA migrates in a gel. The factors are the size of the DNA, the agarose concentration, the conformation of the molecule, the voltage, the presence of ethidium bromide, the type of agarose and the buffer. Ethidium bromide, a fluorescent stain described later, reduces the migration rate by about 15 percent.
3:13 Choose agarose or polyacrylamide and the percentage
The sieving depends on the matrix, so the matrix is the next choice. Agarose is a polysaccharide that comes from red seaweed, mainly the genera Gelidium and Gracilaria. Its polymers associate into bundles, and the gel sets as it cools. The bundles form a network with pores between them, and the DNA threads through the pores. Agarose separates fragments from 100 base pairs to 25 kilobases. Polyacrylamide forms by a free-radical reaction instead of by cooling. You run polyacrylamide as a thin vertical gel at a higher concentration. It suits fragments shorter than 100 base pairs. Pore size falls as the agarose concentration rises. Most agarose gels use 0.5 to 2 percent weight per volume. The concentration is the mass of agarose in grams per 100 mL of buffer. A 1 percent gel holds one gram of agarose in 100 mL of buffer. You pick the percentage from the sizes you need to separate. A 1.5 kilobase PCR product separates well on a 1 percent gel. A 200 base pair product separates well on a 2 percent gel. A 12 kilobase plasmid digest needs about 0.5 percent. One widely reproduced table pairs 0.5 percent with 1 to 20 kilobases, and 2 percent with 0.1 to 2 kilobases. Supplier tables give different numbers for the same percentages, so treat the pairings as a guide. Fragments longer than 25 kilobases do not separate on a standard agarose gel. They need pulsed field electrophoresis, which switches the direction of the field.
4:54 Running buffers, TAE and TBE
With the matrix chosen, the gel needs a running buffer. The buffer carries the current and holds the pH, and you must use the same buffer in the gel and in the tank. Two buffers are common. TAE contains 40 millimolar Tris-acetate and 1 millimolar EDTA. TBE contains 45 millimolar Tris-borate and 1 millimolar EDTA. In both buffers the EDTA inhibits nucleases that need metal ions. TAE works better for separating large fragments, above about 1,500 base pairs, in short runs. TBE buffers more strongly than TAE, so it suits longer runs and fragments below about 5,000 base pairs. The choice also moves the tracking dyes, which matters for deciding where to stop the run.
5:43 Cast a gel
With the buffer chosen, you can cast the gel. The order of the steps is weigh, add buffer, melt, cool, pour and set. Weigh the agarose into a flask that is no more than one third full. Add the running buffer and swirl. Heat the flask in a microwave in 30 second bursts. Swirl between bursts and stop when no particles remain. If you stain inside the gel, add the stain now. The stains are described later. Let the solution cool before you pour. Hot agarose warps the gel tray. The comb forms the wells that hold your samples. A well is a rectangular pocket in the gel. Seal the ends of the tray in a casting apparatus or with tape. Then set the tray on a level bench and place the comb. Pour the gel and let it set at room temperature. Remove the comb steadily when the gel is firm. Keep the gel between 3 and 4 millimeters thick, because a gel thicker than 5 millimeters lets bands diffuse. A level tray and a vertical comb keep the bands straight.
6:48 Loading dye and tracking dyes
With the gel cast, the samples need preparing before they go into the wells. Loading dye does three jobs. It adds density, so the sample sinks into the well. It adds color, so you can see what you are loading. It also contains tracking dyes that show how far the run has gone. A 6X dye is six times its working strength, so you add it at one sixth of the sample volume. For a 30 µL sample, you add 5 µL of 6X dye. Glycerol in the dye gives the density. The dye front is the line you watch during the run. You stop the run when the dye has traveled the distance you want. The dye front gives only a rough size, because its position depends on the gel and the buffer. For example, in a 1 percent gel bromophenol blue runs near 370 base pairs in TAE and near 220 base pairs in TBE. Xylene cyanol runs near 4,160 base pairs in TAE and near 3,030 base pairs in TBE. Orange G runs below 50 base pairs in both buffers. A fragment of the same size as a dye runs with it, and the dye can hide a faint band.
8:01 DNA ladders
The dye gives a rough progress marker, but sizes need a proper reference. A DNA ladder is a mixture of fragments of known sizes, and you load it in one lane beside your samples. Its bands show how far each known size has traveled in this gel. The ladder is your ruler for the rest of the gel. Different ladders contain different sets of sizes. Read the sizes from the sheet that came with the ladder you used. Load the ladder on the same gel as the samples, so that the two run under the same conditions. The ladder lane also shows whether the run itself worked. You compare the sample lanes with the ladder lane.
8:39 Load the gel, set the voltage and run time
With the samples prepared, you can run the gel. Fill the tank with the buffer that you used to make the gel, until it just covers the gel. Load each sample slowly and keep the tip above the bottom of the well. The wells sit at the cathode, which is the negative electrode. DNA carries a negative charge, so it runs from the wells toward the anode. Voltage per centimeter is the voltage divided by the distance between the electrodes. One standard protocol gives 1 to 5 volts per centimeter. The best value depends on the fragment sizes and the buffer, so follow the protocol you are using. A supply set to 100 volts across an electrode spacing of 20 centimeters gives 5 volts per centimeter. Run the gel until the tracking dye has traveled the distance you want. One example gel ran at 100 volts for 1 hour. Stop before the smallest fragments reach the end of the gel. A very high voltage produces heat, which distorts the bands and can denature the sample.
9:40 Stain the gel
The run separates the DNA but does not show it, so you add a stain. A stain that fluoresces when it binds DNA shows the bands. Ethidium bromide intercalates. It inserts between stacked base pairs. Because it binds in proportion to the amount of DNA, a brighter band holds more DNA. Ethidium bromide is also a suspected mutagen and carcinogen. Wear gloves and dispose of the gel and the buffer as hazardous waste under your institution's rules. You can add ethidium bromide to the gel at 0.5 µg/mL. Or you can stain the gel after the run for 15 to 30 minutes. Ethidium bromide bands fluoresce orange under ultraviolet light. Alternatives include SYBR Gold and SYBR Green. Both are sensitive and less toxic than ethidium bromide, and both cost more. Most of these stains work poorly when added to the gel, so you use them as post-stains, which means you apply them after the run. Crystal violet and methyl blue are further alternatives that need no ultraviolet light. Both are less sensitive than ethidium bromide. SYBR Safe, another alternative, absorbs light at about 502 nanometers, so a blue light source excites the stain. The manufacturer reports that SYBR Safe causes fewer mutations than ethidium bromide in the Ames test, a bacterial test for mutagenicity, and that its sensitivity is similar. GelRed is another commercial stain. Its manufacturer reports that it is nonmutagenic and noncytotoxic in the Ames test, so this is also the manufacturer's own report.
11:17 Image the gel
Staining makes the bands fluoresce, and imaging records the pattern. After the run, switch off the supply and remove the gel from the tank. Drain the buffer from the gel. Place the gel on the transilluminator or in the gel documentation system. A gel documentation system holds a camera above the gel and captures the image. Ethidium bromide bands fluoresce orange under ultraviolet light. The camera records the position of each band. Image the gel soon after the run. Bands and stain diffuse during storage, so an old gel gives broader, fainter bands. You measure distances from the well to each band on that image. The image file is the record of the result.
12:01 Read a band size against the ladder
Imaging gives you a picture, and the sizes come from the ladder. In this example the ladder has seven bands, and you measure each distance from the bottom of the well to the center of the band, on the same image. The distances are 10 millimeters for 10 kilobases, 20 for 5 kilobases, 27 for 3 kilobases, 33 for 2 kilobases, 37 for 1.5 kilobases, 43 for 1 kilobase and 53 for 0.5 kilobases. These numbers are illustrative, and they follow the log rule. An unknown band sits at 38 millimeters, between the 1.5 kilobase band and the 1 kilobase band. Take those two ladder bands and work in logarithms. The log of 1,500 is 3.176. The log of 1,000 is 3.000. The two bands are 6 millimeters apart. The unknown is 1 millimeter from the 1.5 kilobase band. The difference between the two logs is 0.176. One sixth of 0.176 is 0.029. Subtract that from 3.176 to get 3.147. Ten to the power 3.147 is about 1,400 base pairs. You can check the answer with a calculator. Plot log size against distance for the ladder bands and draw a straight line through the points. The unknown at 38 millimeters reads about 1.4 kilobases on that line. Interpolating on size directly gives 1.42 kilobases, which is close because the two bands are close together. Use the two nearest bands, and never extrapolate beyond the ends of the ladder. You can measure with a ruler on a printed image. The calculation assumes that the gel ran evenly across its width.
13:50 Check a PCR product
The ladder reading applies directly to checking a product of the polymerase chain reaction, or PCR, which means comparing the size of a band with the size you expect from the primers. You calculate the expected size from the positions of the two primers on the template. A single band at the expected size is the result you want. The no-template control, a reaction set up without template DNA, should give an empty lane. A band in the no-template control shows that contamination reached the reaction. The positive control, a reaction with a template known to work, should show a band. If the positive control is empty, the reaction itself failed. A band near the bottom of the gel can be primer dimers. Primer dimers form when the primers anneal to each other instead of to the template. The expected size tells you which band belongs to your product. Load the ladder in the first lane and the samples in the lanes next to the ladder. Write the lane order down before you load, because the image alone does not record the order.
14:52 Plasmid forms on a gel
A plasmid adds a complication that a linear PCR product does not have, because the conformation of a circular molecule changes how fast it moves. One plasmid preparation can give three bands. The supercoiled form is compact and runs fastest. The linear form runs next. The nicked open circle runs slowest. A nick is a break in one strand of the circle. The linear form runs at its true size. The supercoiled form runs ahead of its true size, so the ladder underestimates the length of the plasmid. To identify the forms, digest an aliquot with an enzyme that cuts the plasmid once. A single cutter is a restriction enzyme with one recognition site on the plasmid. The digest converts the supercoiled and nicked circles into linear molecules of one size. The single band of linear DNA gives the true length of the plasmid. Run the digested sample beside the uncut plasmid. A band in the uncut lane that runs level with the digested sample is the linear form, so the two lanes together identify each form.
15:58 How to approach troubleshooting
Some gels fail, so the remaining sections cover faults. When a gel fails, read the shape of the failure before you change anything. A smear down a lane is a different fault from a missing band. Curved bands, ragged wells, bands that ran off the gel and extra bands each point to their own causes. The next segments take one fault at a time. Each one gives the symptom, the causes, the fixes, and the faults that look similar. Change one thing at a time when you repeat the gel. Keep the ladder lane in the repeat. The two runs then share the same reference. Write down what you changed, so the next gel tests one variable.
16:39 Smeared bands
The first fault to consider is the smear, in which the bands blur into a trail down the lane. One cause is DNA that nucleases have degraded. A nuclease is an enzyme that cuts DNA. Another cause is too much DNA, above 0.1 to 0.2 micrograms per millimeter of well width. A gel thicker than 5 millimeters lets the bands diffuse. Diffusion spreads a band as the molecules move in all directions. Salt or protein in the sample also smears the DNA. Heat from a high voltage does the same. The fixes follow the causes. Load less DNA if the load is the cause. Cast a thinner gel, between 3 and 4 millimeters. Dilute or purify the sample to remove salt and protein before loading. Lower the voltage if the gel is running hot. Three of these faults look similar, so it matters which one you have. Degradation gives a smear that runs down the lane from the well. Overloading gives a smear together with U-shaped bands. Heat gives a smear together with curved bands. To tell them apart, rerun the sample at a lower load. If the smear stays, the DNA is degraded.
17:51 No bands or faint bands
A different failure leaves the ladder lane visible while the sample lanes are empty or faint. One cause is too little DNA, below 0.1 to 0.2 micrograms per millimeter of well width. Degraded DNA gives the same result. A tracking dye of similar size can hide a small band. If the run went too long, the small fragments left the gel. Reversed electrodes drive the DNA backwards, out of the wells and away from the gel. A weak stain or a light source that does not match the stain also gives faint bands. The ladder lane tells you which of these happened. If the ladder is missing as well, suspect the stain, the light source or the electrodes. If only the sample lanes are empty, suspect the amount of DNA or its quality. If only the small bands are missing, the run went too long. Load a known amount of a known sample as a positive control. Check the leads before you switch on the supply. The wells must sit at the negative electrode. Stain the gel again if you are not sure the stain worked.
18:56 Smiling bands and damaged wells
The next fault affects the shape of the bands. In a smiling gel, a row of bands curves like a smile, because uneven heat gives an uneven electric field across the width. Lower the voltage. Use a buffer with a higher capacity, or cool the tank. Level the tray and set the comb vertically. Make sure the agarose has fully dissolved before you pour. Damaged wells are a separate fault. A comb pushed through the bottom of the gel leaves a hole under the well. A comb pulled before the gel has set tears the well. A pipette tip that touches the bottom punctures the well. The two faults look different. Smiling curves the bands in the same way across the whole width of the gel. A damaged well affects one lane. A U-shaped band from overloading also affects one lane. Let the gel set until it is firm to the touch. Pull the comb straight up and steadily. Load the sample slowly, and stop before the tip reaches the bottom.
19:55 Bands off the gel and extra bands
The last two faults concern where bands end up. Bands run off the gel when the run is too long or the voltage is too high. Watch the tracking dye and stop the run before the target size reaches the end. The dye tables tell you which size travels with the dye in your buffer. Extra bands have several causes. A plasmid preparation gives the supercoiled, linear and nicked forms. Fragments with cohesive ends can join into concatemers. A concatemer is two or more fragments joined end to end. A cohesive end is a short single-stranded overhang. Proteins still bound to the DNA after a digestion or a ligation change how the DNA runs. Two different fragments of similar size can run in the same band. Contaminating DNA from another source adds bands. In a PCR lane, primer dimers add a band near the bottom. Compare the positions of the extra bands with the ladder. A band at twice the expected size suggests a concatemer. To test for concatemers or bound protein, add SDS to the loading dye and heat the sample before loading. The heat separates the joined ends. To test for plasmid forms, run a linearized control beside the sample.
21:09 Safety
The faults above cost only time, but the hazards of a gel cost more. The tank connects the mains supply to the buffer, so switch the supply off and unplug the leads before you reach into the tank. Ethidium bromide is a suspected mutagen, so wear gloves and collect the gel and the buffer as hazardous waste. Agarose melted in a microwave can superheat, meaning it stays liquid above its boiling point and then boils over when the flask is disturbed. Heat in short bursts, swirl between them, and never use a sealed container. Ultraviolet light can injure the skin and the eyes. Wear skin and eye protection suited to ultraviolet light, and keep your hands out of the light path. Do not look at the transilluminator without protection. Keep the lid of the gel documentation system closed during imaging. The manufacturer of SYBR Safe reports that blue light damages DNA less than ultraviolet light does. Blue light still needs care.
22:07 Summary and practice question
DNA carries a negative charge on its phosphate backbone, so it moves toward the anode. The gel sorts the fragments by size, and the distance traveled falls as the log of the size rises. You choose the agarose percentage and the buffer for the sizes you need to separate. Loading dye shows the progress of the run, and the ladder gives the sizes. A stain makes the DNA visible, and the imager records the bands. Plasmid forms run at different speeds, so one plasmid can give three bands. You read a failed gel from the shape of the fault. A ladder has a 3 kilobase band at 27 millimeters and a 2 kilobase band at 33 millimeters. An unknown band sits at 30 millimeters. What is the log of its size, and what is its size in base pairs?