Download Restriction Digest of pAMP and pKAN

Restriction Digest of pAMP and pKAN
INTRODUCTION
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, BamH I and Hind III, to digest
two plasmids and electrophoresis to separate the 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 to facilitate gene cloning and protein production
(expression) in bacteria. 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. 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. One contains a gene for ampicillin
resistance, ampr, and the other plasmid contains a gene for kanamycin resistance, kanr. The
plasmids, pAMP and pKAN, were engineered by the DNA Learning Center at Cold Spring
Harbor Laboratory. The pAMP plasmid was engineered from the pUC19 plasmid which
contains ampr and a 1904 bp restriction fragment cut from  DNA. The  restriction fragment
contains both BamH I and Hind III restriction sites. The ligation, or joining, of this fragment into
the pUC19 plasmid gives the engineered plasmid both BamH I and Hind III restriction sites. The
pKAN plasmid was engineered from the pUC19 plasmid and an 1861 bp restriction fragment cut
from the pKC7 plasmid. This pKC7 restriction fragment contains both a kanamycin resistance
gene and BamH I and Hind III restriction sites. Study the following restriction maps of pAMP
and pKAN.
EcoR I
0/4539
Hind III
234
ori
pAMP
4539 bp
r
amp
pKAN
4194 bp
BamH I
1120
r
kan
ori
Hind III
1904
EcoR I
2116
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BamH I
2095
Because Hind III is a specific restriction endonuclease, it will consistently cut DNA
wherever it encounters the six-base 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. These building blocks are oriented in the opposite direction on each strand.
Thus, the two strands that make-up a DNA molecule are said to be “anti-parallel.” For
convenience, we can say that one strand is oriented in a 5’ (“five prime”) to 3’ (“three prime”)
direction while the other strand is oriented 3’ to 5’. Careful examination of the restriction
sequence will reveal that the sequence of nucleotides is a palindrome; that is to say, it reads the
same on both strands when read in a 5’  3’ direction.

5’…………….AAGCTT……………..3’
3’…………….TTCGAA……...……...5’

Therefore, whenever Hind III encounters this six-base sequence, it will cut the DNA helix
between the adjacent adenine bases. This leaves four unpaired bases forming a “sticky end.”
5’…………A 3’
3’…………TTCGA 5’
Sticky end  5’ AGCTT…………3’
 Sticky end
3’ A………….5’
The recognition sequence for BamH I is:

5’…………….GGATCC……………..3’
3’…………….CCTAGG……...……...5’

Can you figure out the 5’3 prime direction “sticky ends” for BamH I?
MATERIALS
Reagents
pAMP (60 ng/μL)
pKAN (60 ng/μL)
Distilled water dH20
Restriction enzyme mix
BamH I + Hind III
2.5x restriction buffer
Equipment and supplies
P-20 micropipette and tips
1.5 mL microfuge tubes
Minicentrifuge
37ºC water bath
Permanent marker
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METHODS
The plasmids pAMP and pKAN have been cloned in overnight cultures of E. coli
(mm294) and purified using a commercial plasmid purification kit. This purification produces a
high yield of supercoiled plasmid, which increases the efficiency of transformations. The
restriction enzymes, BamH I and Hind III, should always be kept in ice to reduce degradation.
Preparing the pAMP and pKAN restriction digest
1. Obtain pAMP, pKAN, 2x buffer and restriction mix from the instructor. Keep these
tubes in ice.
2. Use a marker to label four 1.5 mL microfuge tubes as follows:
pAMP + = pAMP + BamH I and Hind III
pAMP- = uncut pAMP (pAMP without enzymes)
pKAN + = pKAN + BamH I and Hind III
pKAN - = uncut pKAN (pKAN without enzymes)
Include your period and group number on each tube so that you can locate them for
the next lab period.
3. The following reaction matrix summarizes the volumes used to set-up the restriction
digest.
Tube
pKAN
pAMP
2.5x Buffer
dH2O
Enzyme
Mix
Total
Volume
pKAN+
4 μL
-
4 μL
-
2 μL
10 μL
pKAN-
4 μL
-
4 μL
2 μL
-
10 μL
pAMP+
-
4 μL
4 μL
-
2 μL
10 μL
pAMP-
-
4 μL
4 μL
2 μL
-
10 μL
4. Add 4 μL of pAMP to tubes labeled pAMP+ and pAMP-. Change the tip and add 4
μL of pKAN to tubes labeled pKAN+ and pKAN-.
5. Use a fresh tip and add 4 μL of 2.5x restriction buffer to all tubes.
6. Use a fresh tip and add 2 μL of the enzyme mix, containing BamH I and Hind II, to
the tubes labeled pAMP+ and pKAN+ only.
7. Using a fresh tip, add 2 μL of dH2O to the pAMP- and pKAN- tubes. What is the
purpose of these tubes?
8. Cap the tubes and use the minicentrifuge to spin down the reagents.
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9. Place all four tubes into the 37ºC water bath, and incubate for at least 60 minutes.
Following the 60-minute incubation period, the digest can be frozen at -20º C until time
is available for electrophoresis.
Electrophoresis of pAMP and pKAN restriction fragments
INTRODUCTION
It is important at this stage of our experimental procedure that we need to onfirm that
Hind III and BamH I have digested the original plasmids and that we have the correct restriction
fragments. Gel electrophoresis is a procedure commonly used to separate fragments of DNA
according to molecular size or number of base pairs. DNA fragments will migrate through the
agarose maze. DNA, because of the phosphate groups, is negatively charged and will move
towards the positive (red) electrode. Because it is easier for small molecules to move through
the agarose matrix, they will migrate faster than the larger fragments. Picture a group of crosscountry runners that are racing through a dense tropical rain forest. All other factors being equal,
the shorter runners will be able to navigate through the tangle of overhanging vines and dense
foliage faster than the taller runners. So, smaller DNA fragments will move through the tangle
of agarose molecules faster than the longer fragments.
We’ll take all of our plasmid samples, digested, and undigested, and use electrophoresis
to separate these pieces. You might have predicted that your uncut plasmids 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 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 or 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.
We will use a special staining technique that permits us to see the fragments embedded
within the gel, then make a photographic record of your gel to document this important step.
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MATERIALS
Reagents
pAMP (60 ng/μL)
pKAN (60 ng/μL)
DNA size marker (1 kb ladder)
Equipment and supplies
P-20 micropipette and tips
1.5 mL microfuge tubes
Minicentrifuge
Electrophoresis apparatus
UV transilluminator
Poloroid camera
Permanent marker
Agarose (0.8%)
2.5x restriction buffer
Distilled water
1x SB buffer
5x loading dye
Ethidium Bromide
METHODS
1. An 0.8% agarose solution has been prepared and is available in a screw-top culture
tube. The agarose has been pre-measured (about 25 mL) for your gel trays.
2. Be sure the sides of the well tray are in the up position before pouring in the agarose
solution. When the agarose has solidified, about 20 minutes, carefully remove the comb
taking care not to tear the wells. Set the tray into the gel box so that the well-end of the
gel is closest to the negative (black) electrode.
3. Fill the box with 1 x SB buffer to a level that just covers the entire surface of the gel
to a depth of 1-2 mm. Check to see that the gel is covered with buffer and that no
“dimples” appear over the wells. Add more buffer if needed.
4. Determine which samples your team will be loading into the gel: pKAN+ and
pAMP+ and ONE of the following: pKAN-, pAMP-, or DNA marker.
5. Add 2uL of loading dye to each of the tubes containing plasmids (2 or 3, depending
upon your group – be sure to change tips for each tube.
6. If your group is loading the DNA marker, pick it up from your instructor. This sample
already contains the loading dye.
7. Microfuge the tubes.
8. Using a fresh tip for each sample, load 10 uL of each sample into the cells based on
the “map” given in class.
 As you do this, slowly lower the pipette tip below the surface of the buffer directly
over, but not into, the well. Putting the tip into the well can damage the wall of the
well or puncture the bottom of the well. These are not good things to do!
 Use two hands to steady the pipettor. Slowly dispense the sample by pushing to the
second stop of the pipettor. Because of the loading dye, the sample will have a
greater density than the SB buffer. This will allow the sample to sink into the well.
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SB buffer

Important: While holding the button on the second stop, slowly remove the pipette
tip from the gel box. If you’ve loaded your sample correctly, the well will be filled
with a blue colored solution.
9. 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).
10. Turn on the power and set to 130-135 volts.
11. 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.
12. The instructor will remove the gel and stain the gel,
13. Take a photograph of the gel on the UV light box, and include the photo in your lab
write-up.
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