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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, open-circular, 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 thandoes a large, floppy open circle. Therefore, for the same over-all size, supercoiled DNA runs

faster than open-circular DNA. A smaller 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. Thus, an uncut plasmid produces

three bands on a gel, representing each of the conformations. If the plasmid is cut once with a

restriction enzyme, however, the supercoiled and open-circular conformations are all reduced to


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a linear conformation. Following isolation, spontaneous nicks accumulate as a plasmid prep

ages. This can clearly be seen on gels as the proportion of the three conformations change over

time.

The GE lab has a number of different gel boxes in two basic sizes. They are referred toas submarine gels because the slab is completely covered by running buffer. The larger box is

the BRL model H5 (11 x 14 cm gel bed). The gel tray on this box is removable and the gel is

poured outside of the box. There are several versions of the smaller baby gel box. One is the

BRL model H6 (50 x 75 mm gel bed). In the H6, the gel tray is built in to the box. The other

baby gel is manufactured by Carolina Biological. It has a detachable gel tray like the H5.

Each size box has its own best purposes. The H5 is used for most work. The baby gel is used

for quick checks. Its resolution isnt great but the gel runs within 30 to 40 minutes and is very

useful for monitoring longer reactions. The H5 and Carolina 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.


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General Protocol forRunning a Gel

1. Prepare a 1% solution of agarose in 200 ml TBE. One of

the most common beginning mistakes is to make up the

agarose in water instead of TBE. If you do so, your gelswill look very strange. 1% is a standard concentration, but

if you are trying to resolve large fragments of DNA, youmay want to go to a lower concentration. Alternatively, ifyou are trying to resolve small fragments, a higher

concentration would be appropriate.

You may wish to add ethidium bromide at this point. Todo so, add 0.5 g/ml ethidium bromide to both the

running buffer and the agarose. If you dont include thedye here, you will have to stain the gel at the end. 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 ifyou wish to run them farther. However, at the end of

experiment you will end up with lots of ethidium bromide

waste. Even more importantly, ethidium bromide alters

the conformation of the DNA, thereby altering themigration rate. Large fragments contain more ethidium

bromide than smaller fragments, so the rate change would

not be constant over the range of fragments. Dependingon 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.

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 apower 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 TOCLEAN UP THE MESS!

When melted, allow the agarose in a 60o waterbath until it is cool enough to handle. If you pour a gel

while it is too hot, it will warp the plastic gel box,

possibly causing permanent damage.


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3. While the agarose is melting, tightly seal the ends of thegel tray with tape according to the diagram below:

4. Finally, pour 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.

5. When the gel hardens, remove the tape and place the

gel tray into the box. Add buffer to both reservoirs

and cover the gel to a depth of about 2 mm.

6. 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).


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7. Turn on power and run for the appropriate length of

time. The baby gel can run at about 80 volts for

about 40 minutes. The H5 can be run at 110 - 125

volts for about 2 hours, or at 15 volts for an overnight

electrophoresis. Running at greater voltages willresult 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!

9. At the end of the run, shut off the power and

disconnect the electrodes. 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 15 l ofethidium bromide (10 mg/ml). When you add the

ethidium bromide, 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.

Ethidium Bromide is a Powerful Mutagen. Always

Wear Gloves, Glasses, and Lab Coat When

Handling It!

10. Remove the gel from the tray and lay it on the tray

lid. Briefly rinse the gel with water to remove excess

ethidium bromide. Return the ethidium bromide

solution to an empty container. The second and all

subsequent times that you stain gels, you will use

your used ethidium bromide solution. Towards the

end of the term, it may be necessary to freshen the

ethidium bromide. At the end of the quarter, he

ethidium bromide will be collected and de-toxifiedby the instructor for proper disposal.


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11. 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 ofwater for about an hour.

Capturing a Gel With

The BioDoc-It GelDocumentation System

1. Turn on main power switch

2. Turn on Transiluminator


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3. 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 wateron 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 UV-Blocking Gel Viewer door. You can manipulate your gel

by inserting your hands through the side doors.

4. While watching the LCD, rotate the f-stop ring until theimage is bright enough to see on the monitor. (the lower

the f-stop number, the brighter the image will be).

5. Focus the image if necessary.

6. Adjust the zoom as appropriate

7. Fine-adjust the brightness of the image by pressing the

+ or - buttons on the touch pad to brighten or

darken, respectively, the image.

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.

9. 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 limitedand 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#####). Recordthe number for future reference.

10. Print your image by pressing the Print button on the

adjacent thermal printer. This will give you a small, butvery clear image for immediate analysis. For more

detailed analysis, you should work with your recorded

image.


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1. 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 favoriteimage editing software.

2. From the BioDoc-It (from CF card only)

a. Insert the CF cardb, Press Set Up

c. Use the + and - buttons to navigate to the

line READ IMAGES.

d. Use the + and - buttons to navigate to thedesired file.

e. Press Set Up to open the file.

f. Print your image by pressing the Printbutton on the adjacent thermal printer.

3. Remote access to the BioDoc-It memory

a. Point your favorite web browser to

ftp://129.21.156.188 (there is a hotlink to thissite on the Genetic Engineering home page)

b. A username and password are required to log

into the BioDoc-It. Enter biodocit as theusername. Leave the password blank.

c. Open the desired file with your favorite image

processing software.

Accessing a Gel

Image File

d. Note that in order to access images remotely,the BioDoc-It needs to be left on.

Analyzing a Gel 1. Sometimes only a visual analysis is necessary to seewhether bands have changed, disappeared, etc. relative to

controls.

2. To calculate molecular weights, you must first measure

the distance migrated by each band in each lane, and

record this in a table.


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3. Compare your molecular weight standards with the keybelow. 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 yourbands 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 thisband 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 yourstandard graph.


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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 26.

5. 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 thebands.

6. Once you have plotted your standard curve, locate the

distance of your unknown bands, which you have alreadymeasured, on the standard curve. Now you can read the

molecular weight directly off of the log scale.

7. The distance traveled is proportional not only to the sizeof 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, 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. If #1 runs4 cm, #2 will run 2 cm. If #1 runs 3 cm, #2 will run 1.5cm. 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.

9. 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 distancestraveled by all of the other bands and divide each distance

by your standard. Instead of plotting cm traveled, youcan now plot distance traveled relative to the standard. In

this way, you can compare any gel, run at any length oftime.


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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 thenrepeat. 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 afactor 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

placednext to a strip of linear graph graph paper and the set ofvalues 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 cansee that the points fall in the same place on both graphs.

Number Logarithm2 0.3

4 0.68 0.9

20 1.340 1.6

80 1.9

200 2.3

400 2.6800 2.9


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1X TBE Buffer

Final

Concentration

Water (liters) 1.0 2.0 2.5 3.0

Tris Base (grams) 89 mM 10.8 21.6 27.0 16.5

Boric Acid (grams) 89 mM 5.5 11.0 13.6 16.5

disodium EDTA (grams) 2.5 mm 0.93 1.85 2.3 2.8

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