Download Restriction Digest of pAMP and pKAN

Name: _________________________________________________________ Period: _______
Restriction Digest of pAMP and pKAN
There are several methods used to analyze DNA. The purpose of this laboratory is to
introduce one method commonly used to analyze a DNA plasmid (circular, double-stranded
DNA). The protocol uses a mixture of two restriction enzymes, BamHI and HindIII, to digest
(cut) two plasmids and electrophoresis to separate those restriction fragments. DNA that is cut
with restriction enzymes will leave a specific electrophoresis gel pattern. This restriction
fragment pattern should be consistent for any given piece of DNA. Because of the consistency
of cutting, a plasmid can be identified by the pattern of restriction fragments visible in the gel.
Plasmids are circular pieces of DNA that are naturally found in bacterial cells but have
been modified through genetic engineering. Antibiotic resistant genes have been engineered
into these plasmids and function as selectable markers – that is to say, these genes allow us to
select between bacteria that harbor the plasmids from those that do not. If a bacterium carries a
plasmid with an antibiotic resistant gene, the bacterium will be able to grow and reproduce in
the presence of that antibiotic; those bacteria without the plasmid will not be able to grow. For
example, a bacterium that carries a gene to resist the antibiotic kanamycin can be grown in the
presence of kanamycin. If a bacterium does not possess the gene to resist kanamycin it will be
killed by the antibiotic. Thus, antibiotics can be used to select bacteria that are resistant, and
presumably carry a plasmid with the resistant gene, from those bacteria that do not carry the
plasmid.
Two plasmids will be used in this laboratory exercise. pAMP is a plasmid that contains a
gene for ampicillin resistance, ampr. pKAN is the other plasmid and it contains a gene for
kanamycin resistance, kanr. This activity will be divided into two stages:
Stage 1: Plasmid digestion with the help of restriction enzymes
Stage 2: DNA electrophoresis
Stage 1: Plasmid digestion with the help of restriction enzymes
pKAN
pAMP
 The “p” stands for plasmid
 The “p” stands for plasmid
 “KAN” means the plasmid is
 “AMP” means the plasmid is
resistant to the antibiotic
resistant to the antibiotic called
kanamycin
ampicillin
pKAN
5512bp
BamHI
pAMP
4872bp
807bp
r
kan
HindIII
ampr
BamHI
HindIII
377bp
Amgen has measured the size of pKAN to be 5,512 base pairs (bp) in size and pAMP to be
4,872bp in size. If cut properly by restriction enzymes BamHI and HindIII, each plasmid should
be digested into two fragments. Calculate the size of the pKAN and pAMP fragments below.
Small pKAN fragment = __________
Small pAMP fragment = __________
Large pKAN fragment = __________
Large pAMP fragment = __________
HindIII is a restriction enzyme that will consistently cut DNA wherever it encounters the sixbase recognition sequence indicated below. The precise location that is cut is called its
restriction site. The DNA molecule consists of two strands of nucleotide building blocks.
Therefore, whenever HindIII encounters this six-base sequence seen below, it will cut the DNA
helix between the adjacent adenine bases. This leaves four unpaired bases forming a “sticky
end.”
The recognition sequence for BamHI is:
By this stage, you should know that restriction enzymes cut plasmids into fragments. In the first
part of this activity, you will mix plasmid pKAN and plasmid pAMP with restriction enzymes
BamHI and HindIII. If done properly, the restriction enzymes should cut the plasmids into the
fragments calculated above. Follow the directions below very carefully.
Directions:
1. Obtain four microtubes and label their caps: A+, A-, K+, KA+ = contains pAMP + restriction enzymes
A- = contains pAMP but no restriction enzymes
K+ = contains pKAN + restriction enzymes
K- = contains pKAN but no restriction enzymes
2. Also write your group number on the four tubes so they can be identified later.
3. Add the following materials to the tubes labeled A+ and Aa. Add 4μL of pAMP to the A+ tube
b. Add 4μL of pAMP to the A- tube
c. Change tip
d. Add 4μL of buffer to the A+ tube
e. Add 4μL of buffer to the A- tube
f. Change tip
g. Add 2μL of restriction enzymes to the A+ tube
h. Change tip
i. Add 2μL of distilled water to the A- tube
j. Change tip
4. Add the following materials to the tubes labeled K+ and Ka. Add 4μL of pKAN to the K+ tube
b. Add 4μL of pKAN to the K- tube
c. Change tip
d. Add 4μL of buffer to the K+ tube
e. Add 4μL of buffer to the K- tube
f. Change tip
g. Add 2μL of restriction enzymes to the K+ tube
h. Change tip
i. Add 2μL of water to the K- tube
j. Discard tip
5. Close the four microtubes and use the centrifuge to mix all the reagents together. Be sure
your tubes are labeled so they can be identified.
6. Place all four tubes in the 37ºC warm water bath for at least 60 minutes. This will allow
time for the restriction enzymes to cut the plasmids.
7. After an hour, the tubes will be placed inside a freezer until we need them again.
Stage 2: DNA Electrophoresis
At this stage of our experiment we need to confirm that HindIII and Bam H1 have digested
(cut) the plasmids and that we have the correct restriction fragments. Gel electrophoresis is a
procedure commonly used to separate fragments of DNA according to number of base pairs.
Once your plasmid fragments are loaded into the electrophoresis gel, an electric current will be
applied. The plasmid fragments will migrate through the gel with the smaller fragments
traveling a further distance. DNA is negatively charged and will move towards the positive
(red) electrode. This process takes about an hour to complete. When electrophoresis is finished,
we will use a special staining technique that permits us to see the fragments embedded in the
gel. The gel will then be soaked in ethidium bromide, which fluoresces (glows) under
ultraviolet light. During the 20 minute soaking time, the ethidium bromide bonds to the plasmid
fragments. After soaking in ethidium bromide, your gel will be removed and placed on an
ultraviolet light source. Once turned on, the UV light will cause the ethidium bromide that
bonded to the plasmid fragments to glow. We will take a photograph of your gel to document
this important step.
If done properly, the restriction fragments should have been pulled through the electrophoresis
gel and we should see the fragments. But how can we measure them? Marker DNA will also be
added to the gel prior to turning on the electric current. Marker DNA is a collection of known,
precut fragments of DNA. This precut DNA is used like a measuring ruler. The precut Marker
DNA that we will be using will contain 10 fragments of known sizes. The sizes of those 10
fragments can be seen below.
Make a Prediction: According to the sizes calculated, draw the location where you would expect
to find the fragments of pKAN (K+) and pAMP (A+). For now, ignore the K- and A- lanes.
What about K- and A-? Remember that K- does not contain restriction enzymes so therefore it
should be undigested (uncut). Also, A- does not contain restriction enzymes and should be
undigested. Remember that whole pAMP is 4872bp in size, while whole pKAN is 5512bp in
size. Predict in the picture above where you would suspect to find the K- and A- bands when
electrophoresis is complete.
You might have predicted that when loaded into the electrophoresis gel, K- and A- would
produce only a single DNA band; there’s no reason why you would think otherwise. However,
it is likely that two or three bands will appear in the undigested K- and A- plasmid lanes. This
is because plasmids isolated from cells exist in several forms.
One form of plasmid is called “supercoiled.” You can visualize this form by thinking of
a circular piece of plastic tubing that is twisted. This twisting (supercoiling) results in a very
compact molecule; one that will move through the gel very quickly for its size.
A second plasmid form is called a “nicked-circle” or an “open-circle.” Often a plasmid
will experience a break in one of the covalent bonds located in its sugar-phosphate backbone
along one of the two nucleotide strands. Repeated freezing and thawing of the plasmid or other
rough treatment can cause the break. When this break occurs, the tension stored in the
supercoiled plasmid is released as the twisted plasmid unwinds. This circular plasmid form will
not move through the agarose gel as easily as the supercoiled form; although it is the same size,
in terms of base pairs, it will be located closer to the well than the supercoiled form.
The last plasmid form we are likely to see is called the “multimer.” When bacteria
replicate plasmids, the plasmids are often replicated so fast that they end up linked together like
links in a chain. If two plasmids are linked, the multimer will be twice as large as a single
plasmid and will migrate very slowly through the gel. In fact, it will move slower than the
nicked-circle. Your uncut pAMP and pKAN samples then may each have three bands that
appear in the gel. Starting closest to the well, you might observe a multimer, followed by a
nicked-circle band and finally, a fast traveling supercoiled band.
Electrophoresis Directions:
1. Obtain your four microtubes from your teacher. They have been in a freezer since you
last used them.
2. Add a clean tip to your micropipette.
3. Add 2 μL of loading dye to each of the four tubes. Dispose of the tip once finished.
4. Use the centrifuge to mix all the reagents together.
5. Using a fresh tip for each sample, load 10 μL of your sample into the proper
electrophoresis gel lanes as instructed.
6. Notify your teacher when your gel is finished.
Your teacher will perform the following steps:
7. Secure the cover to the electrophoresis box and connect the electrical leads to the power
supply. Be certain that the anode is connected to the anode (red to red) and the cathode is
connected to the cathode (black to black).
8. Turn on the power and set to 130-135 volts.
9. When the loading dye has moved to within 1 cm from the far edge (+ end) of the gel, turn
off the power supply. Unplug the electrical leads from the power supply by grasping the
plugs, not the cords. Remove the cover to the electrophoresis chamber.
10.The instructor will remove the gel and stain the gel with ethidium bromide.
11.Take a photograph of the gel on the UV light box.