DNA RESTRICTION ANALYSIS
In this experiment, DNA from the bacteriophage Lambda (48,502 base pairs in length) is cut
with a variety of restriction enzymes and the resulting fragments are separated using gel
electrophoresis. Three samples of Lambda (phage) DNA are incubated at 37 degrees C,
each with one of the 3 restriction endonuclease enzymes: Pst1, EcoRI, and HindIII. A fourth
sample will be the negative control in that is will be incubated without any endonuclease.
Each of the 3 enzymes recognizes a different sequence of bases on DNA called a
pallindrome , and cuts within it at a specific
site called a "restriction site."
The DNA samples are then loaded into
wells of an agarose gel and
electrophoresed, along with loading dyes
(see procedure below). An electrical field
applied across the gel causes the DNA
fragments in the samples to move from their
origin (a sample well) through the gel matrix
toward the positive electrode. Small DNA
fragments migrate faster than larger ones,
so restriction fragments of differing sizes
separate into distinct bands during electrophoresis. The loading dyes are of 2 different sizes,
corresponding to very small DNA fragments and very large DNA fragments. They can be
seen as the electrophoresis progresses, and they form a 'bracket' in between which the DNA
fragments are moving. Otherwise, one cannot tell how far the DNA fragments have moved
through the agar. The characteristic number and pattern of bands produced by each
restriction enzyme are made visible by staining with a compound that binds to the DNA
molecule--- methylene blue.
OBJECTIVES:
Learn how to cut DNA into fragments with enzymes.
Load and separate DNA fragments by electrophoresis.
Analyze electrophoresis gels.
MATERIAL NEEDED:
restriction buffer
loading dye
ice
37 C degree water bath
Lambda phage DNA
TBE buffer mix
restriction enzymes: Pst1, EcoR1, Hind111
agarose to be poured (in 60 C water bath)
reaction tubes
4 microtubes: clear, violet, green, orange
staining tray
PROCEDURES:
Use of the micropipetter
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Be sure that the amount set on the pipetter is correct.
Place new tip firmly on micropipetter.
Depress plunger to first stop, and hold this position. This step eliminates any air in
tip.
Dip tip into solution to be pipetted, and draw fluid into tip by releasing plunger. Always
touch pipette tip to side of tube to dislodge any small amount stuck to tip. You now
have a sample inside of your pipette tip.
To expel sample, touch pipette tip to inside wall of tube into which you want to empty
sample. This creates a capillary effect which will help draw fluid out of tip.
Slowly depress plunger to first stop and then depress to second stop to blow out last
bit of fluid in tip. Do NOT release plunger before removing tip from fluid in tube.
Otherwise, it will suck fluid back into tip.
When taking a sample, always check for air at the tip. If it is present, put the sample
back and begin again.
Digest DNA with restriction endonucleases (keep all enzymes on ice)
1. Label four 1.5ml tubes, in which you will perform restriction digestion: "P" for Pst1
enzyme, "E" for EcoRI enzyme, "H" for HindIII enzyme, and "L" for Lambda DNA
uncut.
2. Using table below, add reagents to each tube in this order: DNA, restriction buffer,
water, and enzymes last (ask for them). Use a fresh pipette tip for each different
reagent. The amounts are given in microliter (millionths of a liter)
3. Pool and mix reagents by tapping the tube bottom on counter a couple of times (or use
a microcentrifuge).
4. Incubate all reaction tubes for 30 minutes at 37 degrees C.
5. The tubes will go into the freezer until the next period when the electrophoresis will be
done.
color
clear
violet
green
orange
tube code enzyme
L
uncut DNA
P
Pst1
E
EcoRI
H
HindIII
DNA
4
4
4
4
buffer
6
5
5
5
Pst1
--1
-----
EcoRI
----1
---
HindIII
------1
Casting agarose gel
1. Set gel-casting tray into the tray apparatus, screw tight, and insert well-forming comb
into space marked with red line. There is a leveling bubble which can be used to level
the tray (by turning knobs at bottom).
2. Place tray FLAT where agarose can be poured and allowed to set UNDISTURBED.
3. Carefully pour 40ml of agarose solution (liquified in 60 degree C water bath) into
casting tray. Use a toothpick to move any bubbles to edges (this must be done
BEFORE gel hardens).
4. Gel will solidify within 20 minute. Do NOT move tray while agarose is solidifying.
5. Gently remove comb, pulling it straight up and taking care not to rip wells.
6. When solidified, remove the gel tray from the gel-casting tray and place on platform of
electrophoresis box, so that comb is at negative (BLACK) cathode end. The - charged
DNA fragments will migrate towards the + anode end.
7. Fill box with TBE buffer, to level that just covers entire surface of gel by about 2mm.
8. Make certain that sample wells left by comb are completely submerged by buffer.
9. The gel is now ready to load with DNA.
Loading gel with DNA
1. Remove the 4 tubes from the fridge and pulse spin the tubes in a centrifuge or tap the
tubes firmly down on the table top so that all contents go down to the bottom of the
tube.
2. Add 2 ul loading dye to each reaction tube and tap contents of tube on table top.
3. Use pipette to load contents of each reaction tube into a separate well in gel (total of 4
wells). Set your micropipetter on 12 l (that should be the total contents in the
tube).
4. You will need to remember the order of your tubes since there is NO way that the gel
can be marked. Use a clean pipette tip for each different tube.
o Steady pipette over well using 2 hands.
o Expel any air first from pipette tip.
o Dip pipette tip through buffer, positioned over the well, and slowly expel the
mixture (do not punch thru bottom of gel).
5. The loading dye contains sucrose which is heavier than the DNA. It weighs the
mixture down so it will sink into the bottom of the well.
Electrophoresis
1. The electrophoresis chamber top is placed on the chamber, the electrodes connected
to power supply--anode to anode (red-red) and cathode to cathode (black-black).
2. Power supply is turned on and voltage set---120V. The higher the voltage, the faster
the electrophoresis time. In a few minutes, you should begin to notice the loading dye
moving through the gel toward the + pole (anode). We will let it run for about 1 hour.
3. The loading dye will resolve into 2 bands of color. The faster-moving, purplish band
is the dye bromophenol blue: the slower-moving, aqua band is xylene cyanol .
Bromophenol blue migrates through the gel at the same rate as a DNA fragment
approximately 300 base pairs long. Xylene cyanol migrates at the a rate equivalent to
approximately 2,000 base pairs long.
4. After 1 hour, the bromophenol band should be nearing the end of the gel. Turn off the
power supply, disconnect the leads, and remove the top of chamber.
5. Carefully remove the casting tray and keep it horizontal until you are ready to put into
the plastic container. Slide gel easily into staining tray labeled with your group name .
6. Add enough methylene blue DNA stain to cover the gel and place cover on it. They
will sit in the dye overnight.
7. The gels will be washed a couple of times in distilled water (standing for 10-20 min),
and will refrigerate until next lab period when we will look at them.
8. The gels can then be placed on gel support film, which binds the gel and dehydrates it,
if your instructor so chooses.
INTERPRETATION:
Examine your stained gel on a light box, overhead projector, or a UV box.
Which restriction enzyme produced the most restriction sites on the lambda
DNA?
The HindIII digest of lambda DNA yields at least 6 fragments suitable for use as
molecular weight standards for gel electrophoresis. This is called a ladder.
After the sample is ran, the unknown fragments can be compared with the
ladder fragments to determine the approximate size of the unknown DNA bands
by how they match up to the known bands of the ladder.
One can construct a graph plotting
the known base-pair fragments
(the fragments from HINDII
restriction are multiple and of
known size) against the distance
migrated from the well. Once you
have the known fragment sizes
plotted, a straight line is drawn,
and that then enables you to plot
an unknown size fragment and
determine base-pairs.
Example:
An unknown fragment runs to
25mm. A line is drawn up to the
straight line from the X-axis, and
horizontally over to the Y-axis.
The BP value read on the Y-axis is
about 3300bp.
QUESTIONS:
1. What is the source of the DNA being used in this exercise?
2. The speed of the DNA fragment migration correlates directly with the _____ of the
fragment.
3. Why use 3 different restriction enzymes to cut DNA?
4. How can you account for differences in band separation and intensity between your gel
and the ideal gel?
5. Small restriction fragments of nearly the same length will appear as a single band on this
gel, even though it may be run to the very end. Why?
Fall 2011 - Jackie Reynolds, Richland College, BIOL 2421