Agarose Gel Electrophoresis
Agarose gel electrophoresis is one of several physical methods for separating DNA fragments according to
size. 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 several factors that affect migration rate through a gel:
the size and conformation of the DNA, the concentration of agarose, and the 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. Thus small fragments run
more quickly than large fragments, all at a rate proportional to the logarithm of their length. The relationship
of size to migration rate is linear on semi-log paper 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 - 20 kb in length.
Most of the DNA’s that we will be examining are
plasmids. Plasmid DNA can exist in three
conformations: supercoiled, open-circular, and
linear. In vivo, plasmid DNA is a 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 singlestrand nicks. Given the presence of a break in only
one of the strands, the DNA will remain circular, but
the break permits rotation around the phosphodiester
backbone and the supercoils will be released. A
small, compact supercoiled knot of DNA generates
less friction against the agarose matrix than does a
large, floppy open circle. Therefore, for the same
DNA, the supercoiled conformation runs faster than
the open-circular form. If the DNA sustains doublestrand breaks it produces a linear conformation.
Linear DNA runs through a gel end first and thus
generates less friction than open-circular DNA, but
more than supercoiled, and will migrate at a rate
intermediate between the two. 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
21
Electrophoresis
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 during a plasmid prep, much of the DNA may
become nicked, converting supercoiled plasmids to linear conformations. Thus for any particular plasmid
prep, the amount of supercoiled DNA may 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.
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. 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 gels are used for quick checks. Their resolution
isn’t 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
that of a baby gel.
Preparing a Gel
1.
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 beginner’s mistake is
to make up the agarose in water instead of running
buffer. 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 that permits visualization
of a broad range of fragment sizes, but the largest and
smallest fragments are poorly resolved. If you are
22
Electrophoresis
specifically trying to resolve large fragments of DNA,
you may want to use a lower concentration of agarose.
Alternatively, if you are trying to resolve small fragments,
a higher agarose concentration would be appropriate.
2.
Agarose does 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 doesn’t boil over.
IF YOUR AGAROSE BOILS OVER, MAKE SURE TO
CLEAN UP THE MESS!
Running a Gel
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 UVtransparent 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.
4.
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.
5.
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.
1.
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”).
2.
Turn on power and run for the appropriate length of time. For
most applications, running the gel until the tracking dye travels ¾
of the length of the gel is sufficient. 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
23
Electrophoresis
overnight electrophoresis.
There is a heat differential across the surface of a gel; the outside
lanes are cooler the inner lanes and you may see edge effects in
which the outside bands are tilted. It is best to avoid the outside
lanes whenever possible.
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!
3.
At the end of the run, shut off the power and disconnect the
electrodes.
4.
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.
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 restriction map and would like to measure fragment sizes accurately, it is always best to run the
gel in the absence of the dye.
24
Electrophoresis
Visualization With
Stain Added Prior to
Electrophoresis
1.
Agarose is prepared in 200 µl quantities
2.
Gel Red: add 8 µl of Gel Red per 200 ml, and swirl to mix.
Then microwave to melt.
3.
Ethidium Bromide: While agarose is still molten, add 0.5
mg/ml EtBr to both the agarose and the running buffer.
4.
Ethidium Bromide is a Powerful Mutagen. Always Wear
Gloves, Glasses, and Lab Coat When Handling It!
Pour gel when cool. Allow the remainder to solidify. When you
next use the agarose, merely melt it.
Visualization
Following
Electrophoresis
1.
Carefully transfer the gel to a staining tray.
2.
The first time you stain a gel, cover it with about 100 ml of TBE
and add 5 µl of Gel Red OR 15 µl 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.
3.
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.
.
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.
25
Electrophoresis
1X TBE Running Buffer
Final
Concentration
Water (liters)
Tris Base (grams)
Boric Acid (grams)
Disodium EDTA (grams)
1.0
10.8
5.5
0.93
89 mM
89 mM
2.5 mm
Capturing a Gel With The
BioDoc-It Gel
Documentation System
1.
Turn on main power switch
2.
Turn on Transiluminator
26
2.0
21.6
11.0
1.85
2.5
27.0
13.6
2.3
3.0
16.5
16.5
2.8
Electrophoresis
3.
Lay the gel on the Transiluminator (UV is automatically
switched off when main door is open). After you lay your
gel on the Transiluminator, you should slide it to one side
and wipe up the excess water. If there is too much water
on the Transiluminator, your gel will drift out of position
while you are trying to photograph it. You can safely
view your gel under UV by opening the UV-Blocking Gel
Viewer door. You can manipulate your gel by inserting
your gloved hands through the side doors.
4.
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).
5.
Focus the image if necessary.
6.
Twist the zoom ring to adjust the image size 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-It’s
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.
10.
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.
27
Electrophoresis
Accessing a Gel
Image File
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 favorite image
editing software.
2.
3.
4.
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
a.
Point your favorite web browser to
ftp://129.21.156.188 (there is a hotlink to this site on
the Genetic Engineering home page)
b.
The BioDoc-It can also be accessed from an iPad or
other type of tablet. To do so from an iPad (and
probably other tablets as well) you will need an FTP
app.
28
Electrophoresis
c.
You will be asked for a username and password, but
these are not required. Merely press “RETURN”.
d.
Open the desired file with your favorite image
processing software.
e.
Note: in order to access images remotely, the
BioDoc-It needs to be left on.
29
Electrophoresis
Analyzing a Gel
1.
Sometimes only a visual analysis is necessary to see
whether 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.
3.
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 31.
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 the
bands.
6.
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.
7.
A basic knowledge of logarithms is helpful in
understanding how semi-log paper works.
30
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 shorthand expressions of large numbers. In micro- and
molecular biology 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 molecular biology 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, then 2 = 10 0.3010
if log 3 = 0.4771, then 3 = 10 0.4771
if log 4 = 0.6021, then 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 n, the decimal point is
moved n 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 using 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.
31
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 as the
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
When multiplying by logs, the logs are added:
2 x 4 = log 2 + log 4 = 0.3010 + 0.6021 = 0.9031: antilog 0.9031 = 8
2
When dividing by logs, the log of the divisor is subtracted from the log of the dividend:
40 / 8 = log 40 – log 8 = 1.6021 – 0.9031 = 0.6990: antilog 0.6990 = 5
3
When raising a number to a power, the log of the number is multiplied by the exponent:
82 = 2 x log 8 = 2 x (0.9031) = 1.8062: antilog 1.8062 = 64
4
When taking the root of a number, the log is divided by the root:
3
√ 27 = log 27 / 3 = (1.4314) / 3 = 0.4771: antilog 0.4771 = 3
By using this brief review of logarithms and the 4 log rules, you should have no difficulty in mastering
any mathematical problems in molecular biology.
32
Electrophoresis
33
Electrophoresis
Plotting on Semi-Log Paper
The abscissa (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 ordinate (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 are 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
or
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 2 3 4 5 6 7 8 9 10
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 numerical 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
34
Electrophoresis
Relative Migration Distance
Occasionally you may wish to directly compare fragments run on different gels. For example, you wish to
plot 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 by a DNA fragment is proportional
not only to its size, but also to the time that the gel was
allowed to run. Thus the same DNA fragment run on two
different gels will not be directly comparable.
You can directly compare different gels by plotting not
actual distance migrated, but relative distance.
2.
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.
Thus the overall pattern of bands will be the same even if
the distances between bands are expanded or compressed.
If we arbitrarily assign a value of 1.0 to fragment #1, then
fragment #2 will be 0.5.
3.
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 for any length of time.
35