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 DNA’s 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.

20

it has to be melted. The GE lab has a number of different gel boxes in two basic sizes. or “piggy-back” with a second set of combs in the middle. Note. scale up the volumes accordingly. In most cases. Rather.Electrophoresis Agarose gels are referred to as submarine gels because the slab is laid horizontally and is completely covered by running buffer. Alternatively. See the section below about gel visualization. but if you are trying to resolve large fragments of DNA. The smaller boxes typically referred to as “baby gels” have gel beds of approximately 50 x 75 cm. 2. The microwave should be set to “micro cook” for about 2. Agarose will not dissolve. Baby gels”are used for quick checks. a 1% gel is 1 g agarose in 100 ml buffer. One of the most common beginning mistakes is to make up the agarose in water instead of TBE. In this way. That is. a higher concentration would be appropriate. Preparing a Gel 1. your gels will look very strange. Each type of gel box has its own unique way of sealing the gel bed to prevent leakage of the agarose. MAKE SURE TO CLEAN UP THE MESS! 21 . You can. The larger boxes have gel beds of approximately 11 x 14 cm (gel bed).5 minutes at a power setting of 7. twice the number of samples can be run. This can be added at this point. Their resolution isn’t great but the gel runs within 30 to 40 minutes and is very useful for monitoring longer reactions. this is done in a microwave. Please refer to the instructor or TA if you are unsure. or after you finish the running the gel. but the resolution is similar to a baby gel. You should watch it carefully while it is melting so that it doesn’t boil over. At some point you will need to use a UV-fluorescent dye to visualize the DNA on your gel. Gels are prepared as percentage weight/volume solutions. IF YOUR AGAROSE BOILS OVER. The larger boxes can be run either with a single comb at the top of the gel. the gel tray is removable and the gel is poured outside of the box. you may want to go to a lower concentration. If you do so. Thus. Typically. of course. the weight of agarose in grams per 100 ml running buffer. if you are trying to resolve small fragments. 1% is a standard concentration.

You can be sure that your gel is running by checking for bubbles from the electrodes. Insert the comb and allow gel to harden. Turn on power and run for the appropriate length of time. which is very expensive. Gel trays are made of UV-transparent plastic.Electrophoresis 3. shut off the power and disconnect the electrodes. 22 . When the gel hardens. Running a Gel 1. Remember: DNA is negatively charged and runs towards the positive electrode. Please be careful. 4. If you pour the gel while it is too hot. you run the risk of warping and ruining the gel tray. The larger gels can be run at 100 . At the end of the run. Seal the gel bed as appropriate for your box. Running at greater voltages will result in heating of the gel and distortion of the bands. add buffer to both reservoirs and cover the gel to a depth of about 2 mm.80 volts for about 40 minutes.7 mm thick (you will gain a feel for the proper depth once you have done several). or are staining the gel after the run. The black electrode should be closest to your samples and the red electrode farthest (DNA should “run to the red”). it should be cool enough that you can hold it comfortably in your bare hand. or at 15 volts for an overnight electrophoresis. allow the agarose to cool. Caution: Gels Run at High Voltage and Can Deliver Powerful Electric Shocks! 2. taking care not to tear the wells. 5. The procedure depends on whether you are using ethidium bromide or Gel Red to stain your DNA. 3. Very carefully wiggle the comb out of the gel. insert the combs. You should make the gel about 5 . Before you pour your gel. The baby gel can run at about 60 .105 volts for about 2 hours. and whether you added the dye directly to the agarose. When melted. The next step is visualization. Load your gel (for example with a restriction digest) and attach electrodes. and pour the agarose into the tray. 4.

However. In this lab. Allow remainder to solidify. EtBr alters the conformation of the DNA. this may or may not be a problem. add 0. at the end of the experiment you will end up with lots of waste. Return the staining solution to an empty container. nontoxic dyes have been introduced. thereby altering the migration rate. Visualization With Stain Added Prior to Electrophoresis 1. Briefly rinse the gel with water to remove excess stain. Ethidium Bromide: While agarose is still molten. Visualization Following Electrophoresis 1. 4.5 mg/ml EtBr to both the agarose and the running buffer. However. 3. Glasses. Then microwave to melt. 3. Place the tray on a shaker for 20 minutes. so the rate change would not be constant over the range of fragments. When you add the stain. ethidium bromide (EtBr) has been used for this purpose. More recently. 23 . Traditionally. it is always best to run the gel in the absence of the dye. Agarose is prepared in 200 µl quantities Gel Red: add 8 µl of Gel Red per 200 ml. Large fragments contain more EtBr than smaller fragments. 4 Ethidium Bromide is a Powerful Mutagen. cover it with about 100 ml of TBE and add add 5 µl of Gel Red OR 15 ml of EtBr (10 mg/ml). It is sometimes useful to have the dye present while the gel is running because you can always interrupt the run. Some could stick to the gel and cause an unsightly fluorescent spot (usually in the most critical place). take care not to pipet it directly onto the gel. toxic in the case of EtBr. Depending on the experiment. Always Wear Gloves. When you next use the agarose. The first time you stain a gel. Carefully transfer the gel to a staining tray. merely melt it. The remaining stain is still good. 2. if you are trying to generate a restricition map and would like to measure fragment sizes accurately. Even more importantly.Electrophoresis In order to see DNA on a gel. and then continue if you wish to run them farther. and Lab Coat When Handling It! Pour gel when cool. EtBr is mutagenic and must be handled as hazardous waste. check the location of your DNA fragments. and swirl to mix. the gel must first be stained with a dye that binds to DNA and fluoresces under ultraviolet light. Remove the gel from the tray and lay it on the tray lid. 2. we will mostly use Gel Red.

0 21.85 2.Electrophoresis . 4. it may be necessary to “freshen” the stain.6 2.0 13. The second and all subsequent times that you stain gels. obscure some bands. you will use your used staining solution.8 24 .8 5.5 2. if heavy enough.5 0. destaining may help.6 11.3 3.0 10. Later in the term. 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. At the end of the quarter.0 1.5 16.5 27.93 2. but if your bands are faint. Usually it is not necessary to destain the gel. Destaining is accomplished by soaking the gel in an excess of water for about an hour. Destaining: Occasionally the gel absorbs a background of ethidium bromide which could. EtBr will be collected and properly disposed of.0 16. the Gel Red solution will be discarded.

Focus the image if necessary. you should slide it to one side and wipe up the excess water. While watching the LCD. 2. You can safely view your gel under UV by opening the UVBlocking Gel Viewer door. If there is too much water on the transilluminator. the image. 6. the brighter the image will be). your slide will drift out of position while you are trying to photograph. rotate the f-stop ring until the image is bright enough to see on the monitor (the lower the f-stop number. 3. 7. respectively. 5. After you lay your gel on the transilluminator. 4. 25 .Electrophoresis Capturing a Gel With The BioDoc-It Gel Documentation System 1. Turn on main power switch Turn on Transiluminator Lay the gel on the transilluniator (UV is automatically switched off when main door is open). 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. You can manipulate your gel by inserting your gloved hands through the side doors.

Press “Set Up” to open the file. The memory is limited and your image can be quickly overwritten if there is heavy use. c. Transfer the image to the computer. Via E-mail a. b. Press “Set Up” c. remotely). Press “Save” to record the image to the BioDoc-It’s memory. If you insert a CF card. 10. f. it will save to both the internal memory and the card. Open the image directly with your favorite image editing software. you should work with your recorded image. Accessing a Gel Image File 1. Print your image by pressing the “Print” button on the adjacent thermal printer. From the BioDoc-It (from CF card only) a. 2. 3. or printed.Electrophoresis 8. saved. Insert the CF card b. Access your RIT account and email the image to yourself. b. The word “Frz” will display at the bottom of the screen. but very clear image for immediate analysis. This will give you a small. press the “Capture” button. Print your image by pressing the “Print” button on the adjacent thermal printer.” d. When the image is satisfactory. Insert the CF card into a card reader attached to your computer. e. Use the “+” and “-“ buttons to navigate to the desired file. Record the number for future reference. The BioDoc-It will save the image as a TIFF file and assign it a unique number (UVP#####). Use the “+” and “-“ buttons to navigate to the line “READ IMAGES. This will hold the image on screen to be viewed. For more detailed analysis. 4. Remote access to the BioDoc-It memory 9. Access the BioDoc-It via the lab computer (or your own. 26 . Directly from the CF card a.

c. Leave the password blank. the BioDoc-It needs to be left on. b.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.21.Electrophoresis a. Note that in order to access images remotely. Point your favorite web browser to ftp://129. Open the desired file with your favorite image processing software.156. d. Enter “biodocit” as the username. 27 .

For more on plotting logs. the 1 kb band of 1 kb ladder standard is always clear and distinguishable. see page 28. DNA runs in a gel as a function of the logarithm of its molecular weight. then you have most likely misidentified the bands. you must first measure the distance migrated by each band in each lane. etc. disappeared. relative to controls. you must plot the graph of your gel on semi-log paper. 4. A better idea is to match up the bands according to spacing and pattern.Electrophoresis Analyzing a Gel 1. Therefore. on the standard curve. If they do not. you will usually misidentify the bands if you simply count from one end to the other. Once you have identified the bands. You will notice that bands closer to the wells are more compressed than bands farther away. 6. which you have already measured. For example. Once you have plotted your standard curve. Sometimes only a visual analysis is necessary to see whether bands have changed. Thus. 28 . Moreover. bands that are farthest from the wells are indistinct and often missed. Compare your molecular weight standards with the key above. 5. locate the distance of your unknown bands. they should be plotted on the same graph and they should fall on the same curve. If you run two standards. Now you can read the molecular weight directly off of the log scale. 3. enter the sizes onto your table of distances migrated. and record this in a table. 2. find this band on your gel and then count in both directions until you lose confidence in your ability to identify bands. To calculate molecular weights. Now you can plot your standard graph.

not merely tacked on. then.3010. Logarithms. but in practice.4771 if log 4 = 0. are short-hand expressions of large numbers. only two are used routinely. then.301. For a definition of logarithms. really is added to the characteristic. log 2 log 20 log 200 log 2000 = = = = 0. is log 2 + log 1000. The mantissa. The decimal portion is called the mantissa and is the exponent of 10 used to derive the number in question. 102 = 100 or 2 = log 10100 In the following examples: if log 2 = 0.6021. 29 . 3 = 10 0. then. otherwise known as the common logarithm.3010 if log 3 = 0. In microbiology we also have to deal with large numbers and logs makes the task much simpler. or 0. In the following example using base 10. 2 = 10 0. or 2 x 10 -3. The whole number portion is called the characteristic and is used to determine the decimal point. or: y = logax Theoretically any number may be used as a base for a log system. 4 = 10 0.4771.3010 1. consider the equation ay = x where a is a positive number not equal to 1.3010 2. The characteristic is usually one less than the number of decimal places. then. base 10. The log of 2000.6021 Logarithms are usually expressed as a whole number plus a decimal. 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. As the characteristic increases by m. the decimal point is moved m places to the right. is said to be the logarithm of x to the base a. the logs are added.3010 + 3 = 3.3010 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. In microbiology we will be concerned with one of these. 2000 can be rewritten as 2 x 1000.Electrophoresis Our Friend The Logarithm Believe it or not. y. Thus in the examples above.

699".0 should be moved two places to the left to give 0. one can work backwards in a log table and look up the number whose logarithm is X.01. there are four basic rules to remember: 1 2 3 4 When multiplying by logs. Thus the antilog of 0. It is therefore more convenient to have a positive number for the mantissa.3010 . the logs are added When dividing by logs. The log then becomes log 2 + log 10 -2 = 0.02 = log 2 + log 10 -2 + 10 .3010 is the mantissa of 2. the log of the number is multiplied by the exponent When taking the root of a number.10 =0.3010 + 8 – 10 = 8. the antilog of 0. and ask "what number has a mantissa of . or 2 x 10 -2. etc.3010 is 2.3010 + (-2) = -1.699 If you go to a log table.Electrophoresis When computing the log of a decimal. This is called the antilog. This error (you started with 2) is a result of subtracting the characteristic. Many problems are worked by finding the log of the variables and computing the log of the answer. the log is divided by the root By using this brief review of logarithms and the 4 log rules. To find the final answer. say 0. 30 . you will find 5 for an answer.0 and 8 -10 tells you that the characteristic is -2 and that the decimal in 2. the log of the divisor is subtracted from the log of the dividend When raising a number to a power. you should have no difficulty in mastering any mathematical problems in microbiology. Thus log 0. however.3010 + (-2) + 10 . In working with logs.02. To do this we add 10 and then subtract 10 to the characteristic.4771 is 3.02.10 In this form .10 =0. we can express the number as 2 x 0.

Number 2 4 8 20 40 80 200 400 800 Logarithm 0. Thus. The log scale has no zero and the decimal values that you assign to each cycle is arbitrary. for example. the markings are evenly spaced.3 1. On the Y axis. that is.9 31 . The number corresponds to where the logarithm of the number would be plotted if you were plotting on linear paper.6 2. a strip of semi-log paper is placed next to a strip of linear graph paper and the set of values below are plotted. You can see that the points fall in the same place on both graphs.Electrophoresis Plotting on Semi-Log Paper The X axis of semi-log paper is linear. On the semi-log graph the values are plotted according to the numberical value.9 2.3 0. 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. you plot the distance migrated. Each repeat is a cycle and cycles differ from one another by a factor of 10.6 1.9 1.3 2. the numbers are plotted according to their logarithm. Along this axis. the numbers from 1 to 9 vary in spacing and then repeat. On the linear graph.6 0.

#2 will run 2 cm. if you forget a control on one. If we arbitrarily assign a value of 1. You can directly compare different gels by plotting not actual distance migrated.5 cm.0. 8. you can compare any gel. • If #1 runs 5 cm.0 to fragment #1. Thus the same DNA run on two different gels will not be directly comparable. To calculate relative distance. then fragment #2 will be 0. you wish to plat the results of different gels on the same graph. arbitrarily pick one fragment to be your standard. run at any length of time. Measure the distances traveled by all of the other bands and divide each distance by your standard. etc.5 cm. To do this. In this way. When comparing different gels: • If #1 runs 4 cm. 32 . occasionally you might wish to directly compare different gels. #2 will run 1. • If #1 runs 3 cm. for example.5. but relative distance. Or you may wish to calculate a fragment size from a gel with no molecular weight standard. Measure the distance that it has run and set that equal to 1. The distance traveled is proportional not only to the size of the DNA. or would like to compare different digests run on different gels. Plotting Curves by Relative Migration Distance 1. I usually use the 1 kb band of 1 kb ladder standard. or one that was poorly resolved. For example. Relative distance s based on the following argument: Fragment #1 runs twice as fast as fragment #2. you can now plot distance traveled relative to the standard. but also to the time that the gel was allowed to run. Instead of plotting cm traveled. However. 9. you can calculate Relative Migration Distances.Electrophoresis Relative Migration Distance Occasionally you may wish to directly compare fragments run on different gels. #2 will run 2.

Sign up to vote on this title
UsefulNot useful