Agarose Gel Electrophoresis

Agarose gel electrophoresis is one of several physical methods for determining the size of DNA. In this method, DNA is forced to migrate through a highly cross-linked agarose matrix in response to an electric current. In solution, the phosphates on the DNA are negatively charged, and the molecule will therefore migrate to the positive (red) pole. There are three factors that affect migration rate through a gel; size of the DNA, conformation of the DNA, and ionic strength of the running buffer. In this course, we will use only TBE as a running buffer and therefore ionic strength will be constant throughout all of our experiments. Electrophoresis is essentially a sieving process. The larger the fragment of DNA, the more easily will it become entangled in the matrix and, therefore, the more slowly will it migrate. Small fragments, therefore, run more quickly than large fragments at a rate proportional to their size. The relationship of size to migration rate is linear throughout most of the gel, except for the very largest fragments. Large fragments have a more difficult time penetrating the gel and their migration, therefore, does not have a linear relationship to size. The matrix can be adjusted, though, by increasing the concentration of agarose (tighter matrix) or by decreasing it (looser matrix). A standard 1% agarose gel can resolve DNA from 0.2 - 30 kb in length. Most of the DNAs that we will be examining are plasmids. Plasmid DNA can exist in three conformations: supercoiled, opencircular, and linear. In vivo, plasmid DNA is tightly supercoiled circle to enable it to fit inside the cell. Following a careful plasmid prep, most of the DNA will remain supercoiled, but a certain amount will sustain single-strand nicks. Given the presence of a break in only one of the strands, the DNA will remain circular, but the break will permit rotation around the phosphodiester backbone and the supercoils will be released. A small, compact supercoiled knot of DNA sustains less friction against the agarose matrix than does a large, floppy open circle. Therefore, for the same DNA. A smaller over-all size, supercoiled DNA runs faster than opencircular fraction of the DNA sustains double-strand breaks, producing a linear conformation. Linear DNA runs through a gel end first and thus sustains less friction than open-circular DNA, but more than supercoiled. In addition, plasmids within a single cell tend to recombine with one another producing dimers, trimers, tetramers, etc. However, as multimer size increases, individual multimers recombine with themselves to produce smaller multimers. Thus, an uncut plasmid produces a complex banding pattern on a gel. The three bands with the highest mobility represent supercoiled, linear, and open circular plasmids, followed by a complex ladder of multimers. If the plasmid is cut once with a restriction enzyme, however, the supercoiled and open-circular conformations and all of the multimers are reduced to a linear conformation. Depending on the amount of manipulation that is incurred in plasmid prep, much of the DNA may become nicked, converting supercoiled to linear conformations. This for any particular plasmid prep, the amount of supercoiled DNA my be small to nonexistent. Following isolation and storage, spontaneous nicks tend to accumulate as a plasmid prep ages. This can clearly be seen on gels as the proportion of the three conformations change over time.

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Electrophoresis Agarose gels are referred to as submarine gels because the slab is laid horizontally and is completely covered by running buffer. The GE lab has a number of different gel boxes in two basic sizes. The larger boxes have gel beds of approximately 11 x 14 cm (gel bed). The smaller boxes typically referred to as baby gels have gel beds of approximately 50 x 75 cm. In most cases, the gel tray is removable and the gel is poured outside of the box. Each type of gel box has its own unique way of sealing the gel bed to prevent leakage of the agarose. Please refer to the instructor or TA if you are unsure. Baby gelsare used for quick checks. Their resolution isnt great but the gel runs within 30 to 40 minutes and is very useful for monitoring longer reactions. The larger boxes can be run either with a single comb at the top of the gel, or piggy-back with a second set of combs in the middle. In this way, twice the number of samples can be run, but the resolution is similar to a baby gel.

Preparing a Gel

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Gels are prepared as percentage weight/volume solutions. That is, the weight of agarose in grams per 100 ml running buffer. Thus, a 1% gel is 1 g agarose in 100 ml buffer. You can, of course, scale up the volumes accordingly. Note; One of the most common beginning mistakes is to make up the agarose in water instead of TBE. If you do so, your gels will look very strange. At some point you will need to use a UV-fluorescent dye to visualize the DNA on your gel. This can be added at this point, or after you finish the running the gel. See the section below about gel visualization. 1% is a standard concentration, but if you are trying to resolve large fragments of DNA, you may want to go to a lower concentration. Alternatively, if you are trying to resolve small fragments, a higher concentration would be appropriate.

2.

Agarose will not dissolve. Rather, it has to be melted. Typically, this is done in a microwave. The microwave should be set to micro cook for about 2.5 minutes at a power setting of 7. You should watch it carefully while it is melting so that it doesnt boil over. IF YOUR AGAROSE BOILS OVER, MAKE SURE TO CLEAN UP THE MESS!

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Electrophoresis 3. When melted, allow the agarose to cool. Before you pour your gel, it should be cool enough that you can hold it comfortably in your bare hand. Gel trays are made of UV-transparent plastic, which is very expensive. If you pour the gel while it is too hot, you run the risk of warping and ruining the gel tray. Please be careful. Seal the gel bed as appropriate for your box, insert the combs, and pour the agarose into the tray. You should make the gel about 5 - 7 mm thick (you will gain a feel for the proper depth once you have done several). Insert the comb and allow gel to harden. When the gel hardens, add buffer to both reservoirs and cover the gel to a depth of about 2 mm. Very carefully wiggle the comb out of the gel, taking care not to tear the wells.

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Running a Gel

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Load your gel (for example with a restriction digest) and attach electrodes. Remember: DNA is negatively charged and runs towards the positive electrode. The black electrode should be closest to your samples and the red electrode farthest (DNA should run to the red). Turn on power and run for the appropriate length of time. The baby gel can run at about 60 - 80 volts for about 40 minutes. The larger gels can be run at 100 - 105 volts for about 2 hours, or at 15 volts for an overnight electrophoresis. Running at greater voltages will result in heating of the gel and distortion of the bands. You can be sure that your gel is running by checking for bubbles from the electrodes. Caution: Gels Run at High Voltage and Can Deliver Powerful Electric Shocks!

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At the end of the run, shut off the power and disconnect the electrodes. The next step is visualization. The procedure depends on whether you are using ethidium bromide or Gel Red to stain your DNA, and whether you added the dye directly to the agarose, or are staining the gel after the run.

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Electrophoresis In order to see DNA on a gel, the gel must first be stained with a dye that binds to DNA and fluoresces under ultraviolet light. Traditionally, ethidium bromide (EtBr) has been used for this purpose. EtBr is mutagenic and must be handled as hazardous waste. More recently, nontoxic dyes have been introduced. In this lab, we will mostly use Gel Red. It is sometimes useful to have the dye present while the gel is running because you can always interrupt the run, check the location of your DNA fragments, and then continue if you wish to run them farther. However, at the end of the experiment you will end up with lots of waste, toxic in the case of EtBr. Even more importantly, EtBr alters the conformation of the DNA, thereby altering the migration rate. Large fragments contain more EtBr than smaller fragments, so the rate change would not be constant over the range of fragments. Depending on the experiment, this may or may not be a problem. However, if you are trying to generate a restricition map and would like to measure fragment sizes accurately, it is always best to run the gel in the absence of the dye. Visualization With Stain Added Prior to Electrophoresis 1. 2. Agarose is prepared in 200 l quantities Gel Red: add 8 l of Gel Red per 200 ml, and swirl to mix. Then microwave to melt. Ethidium Bromide: While agarose is still molten, add 0.5 mg/ml EtBr to both the agarose and the running buffer. 3. 4. 4 Ethidium Bromide is a Powerful Mutagen. Always Wear Gloves, Glasses, and Lab Coat When Handling It! Pour gel when cool. Allow remainder to solidify. When you next use the agarose, merely melt it. The remaining stain is still good. Carefully transfer the gel to a staining tray. The first time you stain a gel, cover it with about 100 ml of TBE and add add 5 l of Gel Red OR 15 ml of EtBr (10 mg/ml). When you add the stain, take care not to pipet it directly onto the gel. Some could stick to the gel and cause an unsightly fluorescent spot (usually in the most critical place). Place the tray on a shaker for 20 minutes. Remove the gel from the tray and lay it on the tray lid. Return the staining solution to an empty container. Briefly rinse the gel with water to remove excess stain.

Visualization Following Electrophoresis

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Electrophoresis . The second and all subsequent times that you stain gels, you will use your used staining solution. Later in the term, it may be necessary to freshen the stain. At the end of the quarter, the Gel Red solution will be discarded. EtBr will be collected and properly disposed of. 4. Destaining: Occasionally the gel absorbs a background of ethidium bromide which could, if heavy enough, obscure some bands. Usually it is not necessary to destain the gel, but if your bands are faint, destaining may help. Destaining is accomplished by soaking the gel in an excess of water for about an hour.

1X TBE Running Buffer Final Concentration Water (liters) Tris Base (grams) Boric Acid (grams) disodium EDTA (grams) 89 mM 89 mM 2.5 mm 1.0 10.8 5.5 0.93 2.0 21.6 11.0 1.85 2.5 27.0 13.6 2.3 3.0 16.5 16.5 2.8

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Electrophoresis Capturing a Gel With The BioDoc-It Gel Documentation System

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Turn on main power switch Turn on Transiluminator Lay the gel on the transilluniator (UV is automatically switched off when main door is open). After you lay your gel on the transilluminator, you should slide it to one side and wipe up the excess water. If there is too much water on the transilluminator, your slide will drift out of position while you are trying to photograph. You can safely view your gel under UV by opening the UVBlocking Gel Viewer door. You can manipulate your gel by inserting your gloved hands through the side doors. While watching the LCD, rotate the f-stop ring until the image is bright enough to see on the monitor (the lower the f-stop number, the brighter the image will be). Focus the image if necessary. Adjust the zoom as appropriate Fine-adjust the brightness of the image by pressing the + or - buttons on the touch pad to brighten or darken, respectively, the image.

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Electrophoresis 8. When the image is satisfactory, press the Capture button. The word Frz will display at the bottom of the screen. This will hold the image on screen to be viewed, saved, or printed. Press Save to record the image to the BioDoc-Its memory. If you insert a CF card, it will save to both the internal memory and the card. The memory is limited and your image can be quickly overwritten if there is heavy use. The BioDoc-It will save the image as a TIFF file and assign it a unique number (UVP#####). Record the number for future reference. Print your image by pressing the Print button on the adjacent thermal printer. This will give you a small, but very clear image for immediate analysis. For more detailed analysis, you should work with your recorded image. Directly from the CF card a. Insert the CF card into a card reader attached to your computer. b. Open the image directly with your favorite image editing software. From the BioDoc-It (from CF card only) a. Insert the CF card b, Press Set Up c. Use the + and - buttons to navigate to the line READ IMAGES. d. Use the + and - buttons to navigate to the desired file. e. Press Set Up to open the file. f. Print your image by pressing the Print button on the adjacent thermal printer. Via E-mail a. Access the BioDoc-It via the lab computer (or your own, remotely). b. Transfer the image to the computer. c. Access your RIT account and email the image to yourself. Remote access to the BioDoc-It memory

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Accessing a Gel Image File

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Electrophoresis a. b. c. d. Point your favorite web browser to ftp://129.21.156.188 (there is a hotlink to this site on the Genetic Engineering home page) A username and password are required to log into the BioDoc-It. Enter biodocit as the username. Leave the password blank. Open the desired file with your favorite image processing software. Note that in order to access images remotely, the BioDoc-It needs to be left on.

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Electrophoresis Analyzing a Gel 1. Sometimes only a visual analysis is necessary to see whether bands have changed, disappeared, etc. relative to controls. To calculate molecular weights, you must first measure the distance migrated by each band in each lane, and record this in a table. Compare your molecular weight standards with the key above. You will notice that bands closer to the wells are more compressed than bands farther away. Moreover, bands that are farthest from the wells are indistinct and often missed. Thus, you will usually misidentify the bands if you simply count from one end to the other. A better idea is to match up the bands according to spacing and pattern. For example, the 1 kb band of 1 kb ladder standard is always clear and distinguishable. find this band on your gel and then count in both directions until you lose confidence in your ability to identify bands. Once you have identified the bands, enter the sizes onto your table of distances migrated. Now you can plot your standard graph. 4. DNA runs in a gel as a function of the logarithm of its molecular weight. Therefore, you must plot the graph of your gel on semi-log paper. For more on plotting logs, see page 28. If you run two standards, they should be plotted on the same graph and they should fall on the same curve. If they do not, then you have most likely misidentified the bands. Once you have plotted your standard curve, locate the distance of your unknown bands, which you have already measured, on the standard curve. Now you can read the molecular weight directly off of the log scale.

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Electrophoresis Our Friend The Logarithm Believe it or not, logarithms were created to help you and make mathematics easier! Logs were invented in the early part of the seventeenth century to meet the needs of astronomers who had to deal with very large numbers. Logarithms, then, are short-hand expressions of large numbers. In microbiology we also have to deal with large numbers and logs makes the task much simpler. For a definition of logarithms, consider the equation ay = x where a is a positive number not equal to 1. y, then, is said to be the logarithm of x to the base a, or: y = logax Theoretically any number may be used as a base for a log system, but in practice, only two are used routinely. In microbiology we will be concerned with one of these, base 10, otherwise known as the common logarithm. In the following example using base 10, 102 = 100 or 2 = log 10100 In the following examples: if log 2 = 0.3010, 2 = 10 0.3010 if log 3 = 0.4771, 3 = 10 0.4771 if log 4 = 0.6021, 4 = 10 0.6021 Logarithms are usually expressed as a whole number plus a decimal. The decimal portion is called the mantissa and is the exponent of 10 used to derive the number in question. The whole number portion is called the characteristic and is used to determine the decimal point. The characteristic is usually one less than the number of decimal places. As the characteristic increases by m, the decimal point is moved m places to the right. log 2 log 20 log 200 log 2000 = = = = 0.3010 1.3010 2.3010 3.3010

The relationship of the characteristic to the mantissa can be simplified by applying the first of the four log rules (see below): when multiplying by logs, the logs are added. Thus in the examples above, 2000 can be rewritten as 2 x 1000, or 2 x 10 -3. The log of 2000, then, is log 2 + log 1000, or 0.3010 + 3 = 3.301. The mantissa, then; really is added to the characteristic, not merely tacked on.

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Electrophoresis When computing the log of a decimal, say 0.02, we can express the number as 2 x 0.01, or 2 x 10 -2. The log then becomes log 2 + log 10 -2 = 0.3010 + (-2) = -1.699 If you go to a log table, however, and ask "what number has a mantissa of .699", you will find 5 for an answer. This error (you started with 2) is a result of subtracting the characteristic. It is therefore more convenient to have a positive number for the mantissa. To do this we add 10 and then subtract 10 to the characteristic. Thus log 0.02 = log 2 + log 10 -2 + 10 - 10 =0.3010 + (-2) + 10 - 10 =0.3010 + 8 10 = 8.3010 - 10 In this form .3010 is the mantissa of 2.0 and 8 -10 tells you that the characteristic is -2 and that the decimal in 2.0 should be moved two places to the left to give 0.02. Many problems are worked by finding the log of the variables and computing the log of the answer. To find the final answer, one can work backwards in a log table and look up the number whose logarithm is X. This is called the antilog. Thus the antilog of 0.3010 is 2, the antilog of 0.4771 is 3, etc. In working with logs, there are four basic rules to remember: 1 2 3 4 When multiplying by logs, the logs are added When dividing by logs, the log of the divisor is subtracted from the log of the dividend When raising a number to a power, the log of the number is multiplied by the exponent When taking the root of a number, the log is divided by the root

By using this brief review of logarithms and the 4 log rules, you should have no difficulty in mastering any mathematical problems in microbiology.

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Electrophoresis Plotting on Semi-Log Paper The X axis of semi-log paper is linear, that is, the markings are evenly spaced. Along this axis, you plot the distance migrated. On the Y axis, the numbers from 1 to 9 vary in spacing and then repeat. The number corresponds to where the logarithm of the number would be plotted if you were plotting on linear paper. Each repeat is a cycle and cycles differ from one another by a factor of 10. The log scale has no zero and the decimal values that you assign to each cycle is arbitrary. Thus, for example, two consecutive cycles could read: 1 2 3 4 5 6 7 8 9 10 20 30 40 50 60 70 80 90 100 In the example to the right, a strip of semi-log paper is placed next to a strip of linear graph paper and the set of values below are plotted. On the semi-log graph the values are plotted according to the numberical value. On the linear graph, the numbers are plotted according to their logarithm. You can see that the points fall in the same place on both graphs. Number 2 4 8 20 40 80 200 400 800 Logarithm 0.3 0.6 0.9 1.3 1.6 1.9 2.3 2.6 2.9

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Electrophoresis Relative Migration Distance Occasionally you may wish to directly compare fragments run on different gels. For example, you wish to plat the results of different gels on the same graph. Or you may wish to calculate a fragment size from a gel with no molecular weight standard, or one that was poorly resolved. To do this, you can calculate Relative Migration Distances. Plotting Curves by Relative Migration Distance 1. The distance traveled is proportional not only to the size of the DNA, but also to the time that the gel was allowed to run. Thus the same DNA run on two different gels will not be directly comparable. However, occasionally you might wish to directly compare different gels, for example, if you forget a control on one, or would like to compare different digests run on different gels. You can directly compare different gels by plotting not actual distance migrated, but relative distance. 8. Relative distance s based on the following argument: Fragment #1 runs twice as fast as fragment #2. When comparing different gels: If #1 runs 4 cm, #2 will run 2 cm. If #1 runs 3 cm, #2 will run 1.5 cm. If #1 runs 5 cm, #2 will run 2.5 cm, etc. If we arbitrarily assign a value of 1.0 to fragment #1, then fragment #2 will be 0.5. To calculate relative distance, arbitrarily pick one fragment to be your standard. I usually use the 1 kb band of 1 kb ladder standard. Measure the distance that it has run and set that equal to 1.0. Measure the distances traveled by all of the other bands and divide each distance by your standard. Instead of plotting cm traveled, you can now plot distance traveled relative to the standard. In this way, you can compare any gel, run at any length of time.

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