8/8/2019 15 electrophoresis
1/12
17
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
8/8/2019 15 electrophoresis
2/12
Electrophoresis
18
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.
8/8/2019 15 electrophoresis
3/12
Electrophoresis
19
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.
8/8/2019 15 electrophoresis
4/12
Electrophoresis
20
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).
8/8/2019 15 electrophoresis
5/12
Electrophoresis
21
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.
8/8/2019 15 electrophoresis
6/12
Electrophoresis
22
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
8/8/2019 15 electrophoresis
7/12
Electrophoresis
23
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.
8/8/2019 15 electrophoresis
8/12
Electrophoresis
24
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.
8/8/2019 15 electrophoresis
9/12
Electrophoresis
25
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.
8/8/2019 15 electrophoresis
10/12
Electrophoresis
26
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.
8/8/2019 15 electrophoresis
11/12
Electrophoresis
27
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
8/8/2019 15 electrophoresis
12/12
Electrophoresis
28
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
Top Related