Download Restriction Enzyme Digest and Plasmid mapping

Pre-Lab E 5: Restriction Enzyme Digest and Plasmid mapping (10 pts)
Name: _______________________________; Lab Section: _______________; Grade: _______
1. Describe the function of DNA ladder (1 pts).
2.
Describe the function of loading dye (1 pt).
3. DNA fragments are __________ charged, they will be drawn toward the ________ electrode
(1 pt).
4. Which one is not the essential component of a restriction digest reaction? (1 pt)
a) DNA plasmid
b) Appropriate buffer (10X)
c) Loading dye
d) Restriction Enzyme
5. What is the correct amount of agarose to make 55 ml of 0.75 % agarose gel? (show
calculation and unit, 2 pt.)
6. The plasmid size of pGLO is 5371 bp and the restriction sites for for PstI are at 2106 and
3181. Calculate to predict the sizes of fragments when pGLO is digested with PstI. (1pt)
7. Short Answer: How is DNA visualized on agarose gel? (include reagents and equipments in
your answer) (2 pts)
8. True or False: (1 pt, correct the errors to obtain full credits)
________ . In general, restriction sites are palindromic, meaning the sequence of bases reads
the same forwards as it does backwards on the same DNA strand.
Biol 2281, Spring 2016
E 5: Restriction Enzyme Digest and Plasmid Mapping
Experiment 5: Restriction Enzyme Digest and Plasmid Mapping
Objectives: By the end of this lab, you will be able to:
 Understand the use of restriction enzymes as biotechnology tools
 Become familiar with the principles and techniques of agarose gel electrophoresis
 Estimate DNA fragments sizes from agarose gel data
 Use a restriction map to predict how many fragments will be produced in a given restriction
digest.
Note:
 The introduction was adapted from Restriction Digest and Analysis of Lambda DNA Kit, Bio-Rad
Laboratories, Inc. Duplication of any part of the document is permitted for classroom use only.
Introduction
This excercise introduces you to some important principles of genetic engineering. Specifically, the
functions of restriction enzymes and their use as molecular biology tools will be stressed. Using
agarose gel electrophoresis, you will examine the digestion patterns and determine the sizes of
unknown DNA fragments. Restriction enzymes were a catalyst for the molecular biology revolution,
and now hundreds of such enzymes are commercially available. In this investigation, the restriction
enzymes EcoRV, and Pst I will be used to digest a plasmid, a small circular piece of DNA. Gel
electrophoresis will be employed to separate the resulting DNA fragments, and ethidium bromide will
be used to stain the DNA fragments for visualization.
Restriction Enzymes
The ability to cut and paste, or cleave and ligate, a functional piece of DNA predictably and precisely
is what enables biotechnologists to recombine DNA molecules. This is termed recombinant DNA
technology. The first step in DNA splicing is to locate a specific gene of interest on a chromosome. A
restriction enzyme is then used to cut out the targeted gene from the rest of the chromosome. This
same enzyme is also used to cut the DNA of the recipient into which the fragment will be inserted.
Restriction enzymes are proteins that cut DNA at specific sites. Restriction enzymes, also known as
restriction endonucleases, recognize specific sequences of DNA base pairs and cut, or chemically
separate, DNA at that specific arrangement of base pairs. They were first identified in and isolated
from bacteria that use them as a natural defense mechanism to cut up the invading DNA of
bacteriophages — viruses that infect bacteria. Any foreign DNA encountering a restriction enzyme
will be digested, or cut into many fragments, and rendered ineffective. These enzymes in bacteria make
up the first biological immune system. Each restriction enzyme is named after the bacterium from
which it is isolated. For example:
EcoRI = The first restriction enzyme isolated from Escherichia coli bacteria
EcoRV = The fifth restriction enzyme isolated from Escherichia coli bacteria
PstI = The first restriction enzyme isolated from Providencia stuartii bacteria
Each restriction enzyme recognizes a specific nucleotide sequence in the DNA, called a restriction site,
and cuts the DNA molecule at only that specific sequence. Many restriction enzymes leave a short
length of unpaired bases, called a “sticky” end or “cohesive” end, at the DNA site where they cut,
whereas other restriction enzymes make a cut across both strands creating double-stranded DNA
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Biol 2281, Spring 2016
E 5: Restriction Enzyme Digest and Plasmid Mapping
fragments with “blunt” ends. In general, restriction sites are palindromic, meaning the sequence of
bases reads the same forwards as it does backwards on the opposite DNA strand. For example, here is
a list of enzymes and the sites where they cut:
EcoRI 5’G A-A-T-T-C 3’
3’ C-T-T-A-A G 5’
EcoRV 5’G-A-T A-T-C 3’
3’ C-T-A T-A-G 5’
PstI 5’C-T-G-C-A G 3’
3’G A-C-G-T-C5’
Setting up a simple restriction digest requires four mandatory ingredients:
•
DNA: DNA that is free from contaminants such as phenol or ethanol. Excessive salt will also
interfere with digestion by many enzymes.
•
An appropriate buffer: different enzymes cut optimally in different buffer systems, due to
differing preferences for ionic strength and the type of cations.
•
The restriction enzyme
•
Deionized water: Specific amount of water is required to make up the final volume for a digest
reaction.
In this investigation, students observe the effects of two restriction enzymes on pGLO plasmid DNA.
pGLO plasmid DNA is 5,371 base pairs, each restriction enzyme will cut the DNA one or several
times and generate restriction fragments of different sizes. In this activity, three separate samples of
plasmid DNA will be cut using two different restriction enzymes and the combination of them. Each
sample produces DNA fragments whose sizes can be estimated on an agarose gel electrophoresis.
Electrophoretic Analysis of Restriction Fragments
If a specific restriction site occurs in more than one location on a DNA molecule, a restriction enzyme
will make a cut at each of those sites, resulting in multiple fragments of DNA. Therefore, if a given
piece of linear DNA is cut with a restriction enzyme whose specific recognition sequence is found at
five different locations on the DNA molecule, the result will be six fragments of different lengths. The
length of each fragment will depend upon the location of restriction sites on the DNA molecule.
A DNA fragment that has been cut with restriction enzymes can be separated using a process known as
agarose gel electrophoresis. The term electrophoresis means to carry with electricity. Agarose gel
electrophoresis separates DNA fragments mainly by size. DNA fragments are loaded into an agarose
gel slab, which is placed into a chamber filled with a conductive buffer solution. A direct current is
passed between wire electrodes at each end of the chamber. Since DNA fragments are negatively
charged, they will be drawn toward the positive pole (anode) when placed in an electric field. The
matrix of the agarose gel acts as a molecular sieve through which smaller DNA fragments can move
more easily than larger ones. Therefore, the rate at which a DNA fragment migrates through the gel is
inversely proportional to the log of the molecular weight (size or length). Over a period of time,
smaller DNA fragments will travel farther than larger ones. Fragments of the same size stay together
and migrate in single bands of DNA. A 500 bp ladder containing 16 bands in 500bp increments (the
smallest one is 500 bp) will be used to estimate the sizes of DNA fragments on a 0.8% - 1.0% agarose
gel.
Gels of 0.8-1.0% (w/v, 1 gram of agarose in 100 ml running buffer) agarose will separate fragment
sizes ranging from around 500 base pairs (bp) to around 10,000 bp (or 10 kilobases, kb). Large
fragments (e.g., >10 kb) will be better separated in lower percentage agarose gel, such as a 0.5% gel.
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Biol 2281, Spring 2016
E 5: Restriction Enzyme Digest and Plasmid Mapping
On the other hand, separation of very small (less than 1 kb) can be better achieved in higher percentage
agarose gel, such as a 1.5-2% gel.
Making DNA Visible
DNA is colorless so DNA fragments in the gel cannot be seen during electrophoresis. The loading dye
(or loading buffer) does not stain the DNA itself but makes it easier to load the gels and monitor the
progress of the DNA electrophoresis. Loading buffer usually contains something dense (e.g. glycerol)
to allow the sample to "fall" into the sample wells. It also contains one or two tracking dyes, which
migrate in the gel and allow visual monitoring of how far the electrophoresis has proceeded. The dye
fronts migrate toward the positive end of the gel, just like the DNA fragments. The “faster” dye,
Bromophenol blue, comigrates with DNA fragments of approximately 300 bp, while the “slower” dye,
Xylene cyanol, comigrates with DNA fragments approximately 9 kb in size. Visualization of the DNA
sample requires the use of a transilluminator equipped with a UV lamp. A mid-wavelength (260-369
nm) UV lamp emits light in the optimum range for viewing ethidium bromide-stained gels. You will
view the gels while wearing safety glasses. This fluorescent dye intercalates between bases of DNA
and RNA It is often incorporated into the gel so that staining occurs during electrophoresis, but the gel
can also be stained after electrophoresis by soaking in a dilute solution of ethidium bromide (EB). You
must wear disposable gloves during the lab because EB is a mutagen and a suspected carcinogen. You
can compare the DNA restriction patterns of the different samples of DNA when the bands are visible
using UV lamp.
In addition to electrophoresis, most of the procedures in this exercise employ microchemical
techniques; i.e., using very small amounts of reagents such as DNA and enzymes. In research
laboratory, many reactions of restriction digest require the addition of only one microliter (1 µl, one
one-millionth of a liter) of some components, so it is essential that you become familiar with
measuring small volumes accurately and reproducibly.
Procedures
Materials and Equipments
1) each work station ( for 2 students)
a.
b.
c.
d.
e.
f.
g.
h.
i.
4x loading dye (next to water bath)
A yellow box of pipet tips
micropipetter: p20
a microcentrifuge tube rack
marker
Three microcentrifuge tubes
A tube containing 30 µl of pGLO plasmid (0.03 µg/µl)
One white container for used micropipette tips
diluted EcoRV and PstI restriction enzymes in ice buckets at sink area
2) every group (4 students will share one gel)
a.
b.
c.
d.
e.
f.
g.
Agarose (next to the balance)
125 ml flask for melting agarose
50 ml cylinder (near the sink)
400 ml of 0.5XTBE buffer (near the sink)
Gel apparatus
Power supply
10-teeth comb
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Biol 2281, Spring 2016
E 5: Restriction Enzyme Digest and Plasmid Mapping
Note: the gel apparatus comes as a set, i.e. the lid matches the tank. Clean the gel apparatus and slide the
lid back onto the tank to prevent mismatches!
1. Label three microcentrifuge tubes as follows:
P (PstI digest)
E (EcoRV digest)
D (double digest of PstI and EcoRV, two enzymes pre-mixed)
a) Transfer 10µl of pGLO plasmid to each tube. (check the volume setting on micropipette)
b) Restriction enzymes have been pre-mixed with buffer by instructor and placed in ice buckets near front
sink. Using a red micropipette and a fresh tip for each tube, add 5 µl of diluted restriction enzymes
according to the list above.
c) Flick the tubes to mix well. Spin the tubes for 5 seconds in the microcentrifuge. The tubes should be
placed in a balanced configuration. Do not open the lid if the motor is still running.
d) Incubate the tubes at 37ºC water bath for 50 minutes.
* 1 unit of restriction enzyme activity is defined as the amount of enzyme required to produce a complete
digest on 1µg of substrate DNA in 60 minutes at the appropriate assay temperature in a 50µl reaction
volume.
2.
[one gel per 4 students] Assemble the gel apparatus using a ten slot comb.
a) Place gel casting tray on the top of gel support deck, the open ends of the tray should be placed next
to the sides of the chamber. Insert the comb into the end slot.
b) Wear disposable gloves to prepare 50 ml of the melted 0.8% ( w/v, 0.8 gram/ 100 ml buffer) agarose
solution . The solution contains _____ g of agarose and 50 ml of 0.5X TBE buffer (4.5mM Tris,
4.5mM boric acid, 0.1 mM EDTA, pH 8.3). Boil the buffer and dissolve agarose completely using
microwave with 30 seconds intervals, the suspension needs to be completely clear.
c) Ask TA to add 1µl ethidium bromide to your melted gel solution. Pour the gel solution to the casting
tray. Rinse small flask with water.
d) Demonstrations:
e) After the solidification has occurred, lift the casting tray and rotate it 90 degree. Rinse the comb
with water. Under most conditions, DNA has a negative charge caused by the phosphate groups on
the outer surface of the molecule. Negatively charged DNA molecules will migrate toward the
positive electrode during electrophoresis. You should place the end of gel with wells near
_________(cathode [black] or anode [red]).
f) Slowly fill the electrophoresis chamber with the 0.5X TBE buffer until the gel is completely
submerged. The buffer should be about 3-4 mm above the gel surface.
3. Retrieve your thee digested samples from the 37ºC water bath. Obtain one tube of 4X TBE loading dye
(0.04% bromophenol blue, 20% glycerol, 0.04% xylene cyanol) for two students .
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Biol 2281, Spring 2016
E 5: Restriction Enzyme Digest and Plasmid Mapping
4. (Your bench) Using a fresh tip for each digested sample, add 5 µl of 4X TBE loading dye. Flick the tubes to
mix well and spin for 5 second in the microfuge.
5. Set micropipette (p20) at 20 µl, load your three samples with fresh tips in the appropriate lanes following gel
diagram Fig.1 as a guide. Do not leave space between each sample.
6. Ask TA to load 5 µl of the 500bp DNA ladder (Bio-rad) and uncut pGLO plasmid in the middle lanes. The
DNA ladder contains 16 bands in 500bp increments; the smallest one is 500 bp.
7. When the samples have been loaded, set the power supply to 130V. Run the bromophenol blue dye to within
2 cm of the positive electrode end of the gel (35 minutes approximately). Predict the sizes of fragments in
base pairs based on the information in Fig 3.
8. Visualize the DNA bands on a UV light box. Wear proper eye protection. You may trace the band pattern on
a piece of plastic wrap overlaying the gel or take a picture using a digital camera.
Note: The migration of DNA molecules in agarose gels is roughly proportional to the inverse of the log of
their molecular weight (corresponding to the size of the fragments). The size of the DNA fragments can be
determined by comparing the migration distances (mm) to the standard curve (log bp vs. migration distance)
of the marker if the electrophoresis is run longer to achieve better separation. In this exercise, the length of
DNA fragments will be estimated based on the sizes of marker bands and the information provided by your
instructor.
Cleaning Up:
1.
2.
3.
4.
Return the 4X loading dye tube to the tube rack next to water bath.
Dispose the tips, sample tubes into big biohazard trashcan.
Transfer the gel running buffer into the recycling bucket.
Clean up the tank, the comb, the gel tray with tap water. Do not dry inside of gel box. Place the tray
inside the tank and make sure the lid matches the tank.
5. Refill yellow tip box.
6. Wipe your bench with 70% ethanol (clear top squirt bottle).
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Biol 2281, Spring 2016
E 5: Restriction Enzyme Digest and Plasmid Mapping
wells
PstI
Double
EcoRV
Uncut
pGLO
ladder
EcoRV
Double
PstI
____
____
2000bp
1500bp
1000bp
500bp
Fig. 1: gel diagram
Fig. 2: 500 bp DNA ladder
5371 bp
Fig. 3: The map of pGLO : The restriction site for EcoRV is at 386 and the sites for PstI are at 2106
and 3181)
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Biol 2281, Spring 2016
E 5: Restriction Enzyme Digest and Plasmid Mapping
Post Lab Report (20 pts total): Include questions and a title page in your report (1 pt).
1. The restriction digest map of pUC19 is shown in Figure 4. Suppose you performed restriction
digestions of pUC19 by the various enzymes listed in Table 1 (each row represents a separate
digestion),
1) predict the sizes of fragments in base pairs and write your answers in Table 1(1pt);
2) Label the positions of the wells (1pt), label the lanes with the enzymes used (2pt). Include a lane
with 500 bp DNA ladder (1pt, Fig 2). Draw the corresponding DNA fragments on the gel
diagram, and label the size of each DNA fragment (2pts). Label the cathode and anode (1pt).
0
EcoR I 396
Ava II 2059
pUC19
Ava II 1837
2686 bp
Bsey I 1110
Figure 4. Restriction map of pUC 19, a 2,686 base pair plasmid. The number after each restriction
enzyme name indicates at which base pair the DNA is cut by that enzyme.
Table 1.
Enzymes
Fragments produced
(bp)
EcoRI
Bsey I
Ava II
EcoR1 + Bsey I
EcoR1 + Ava II
Bsey I + Ava II
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Biol 2281, Spring 2016
E 5: Restriction Enzyme Digest and Plasmid Mapping
2. Print the photograph of your gel from eLearning. Label the wells, the lanes with the enzymes
used (2pt). A 500 bp ladder containing 16 bands in 500bp increments (the smallest one is 500 bp)
was used to estimate the sizes of DNA fragments on your gel. Note the sizes of the four smallest
DNA fragments in the molecular ruler/ladder (1pt). Estimate and label the size of each fragment
in each restriction digest sample (1pt). Calculate the sizes of fragments in base pairs based on
the information provided in Fig 3 and label the size for each band (one set) (2pts). Label the
cathode and the anode (1 pt).
3. Visit the New England Biolabs website (www.neb.com) and click on “NEB CUTTER” at the
bottom of the screen. Open the file of pGLO sequence found at eLearning. Copy and paste the
sequence into NEBcutter. Make sure the sequence doesn’t have linebreaks in it. Type “pGLOyour name-BIOL2281” in “Name of sequence”. Select “the sequence is circular”. Select NEB
enzymes and click submit. You will get the restriction map for pGLO.
Then click on Custom Digest under Main Options. Choose EcoRV, PstI. Select “Digest”. It
will display the restriction map with just these enzymes. Click “view gel” under “Main options”.
Print the page with a virtual gel and the list of fragments. Your name should be displayed on the
title of the print-out (2pts).
On the page of “Custom Digest”, five of the open reading frames are identified on the plasmid
map. Click on the curved bars of c. Click “Blast this sequence at NCBI” under the protein
sequence and find out the name of the protein superfamily (1pt). Repeat the search steps for
curved bar of b and find out the name of the protein superfamily (1pt).
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BIOL2281 E5
Restriction Enzyme Digest
Experiment 5: Restriction
Enzyme Digest and Plasmid
Mapping



Restriction Enzyme Digest
Plasmid Mapping
Gel electrophoresis

A strategy for obtaining fragments of DNA

Restriction enzymes cleave segments of DNA from
the genome of various types of cells or fragment
DNA obtained from other sources

Restriction fragments of DNA from different sources
can be used to synthesize a recombinant DNA
1
2
Types of nucleases

Nucleases: catalyze the hydrolysis of phosphodiesters in
nucleic acids


Exonucleases: from one end of a polynucleotide chain
Endonucleases: at various sites within a polynucleotide
chain


Restriction Endonucleases (Enzymes):
synthesized in some bacteria to protect against viral
attack by destroying foreign DNA
How do they protect their own DNA? The host cell modify the bases
of potential restriction sequences by methylation of adenine or
cytosine
DNA cloning: The new plasmid can be introduced into bacterial
cells that can produce many copies of the inserted DNA.
3
4
http://www.accessexcellence.org/RC/VL/GG/inserting.html
Restriction recognition sequences
Nuclease cleavage sites


Restriction Enzymes (RE) cleaves the DNA within or near to the
specific recognition sequences
the nucleotide sequences recognized by more than 200 RE can be
classified as:
a. Tetra- or hexapalindromic sequences:
they are the same sequences when read in the
5' --->
> 3' di
direction
ti off b
both
th strands.
t d
Dpn I 5’GATC3’
EcoR I 5’GAATTC3’
3’CTAG5’
3’CTTAAG5’
b. Pentanucleotide sequences.
c. Sequences of longer extention with internal (N) sequences.
Xmn I (GAANNNNTTC)
d. Nonpalindromic sequences.
5
Dr. Wenju Lin, BIOL2281_Spring 2016
(Nawin Mishra,2002)
6
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BIOL2281 E5
Sticky and Blunt ends
7
8
Figure 8.3
Essential components of a
restriction digest reaction
Microscale

DNA plasmid

Appropriate buffer (10X)

dH2O

Enzyme (unit/µl) : the volume of enzyme should not be
included in final reaction volume
The volumes and tools are adapted to microscale
when we work with DNA

1 ml = 1000 µl (1000 microlitres)
1 mg = 1000 µg (1000 micrograms)

Microcentrifuge tubes
0.25 ml to 2.0 ml
Made of Polypropylene
With Flat tops for writing on
With Graduations, and writing area on the
side of the tube.
One unit of restriction enzyme activity is defined as the amount of enzyme
required to produce a complete digest on 1µg of substrate DNA in 60
minutes at the appropriate assay temperature in a 50µl reaction
volume. Most restriction enzyme reactions are incubated at 37°C
for one hour.
9
Microcentrifuge


Electrophoresis

Use centrifugal force to separate
lighter (rich medium) and heavier
substances (cells).
Spin tubes to ensure efficient usage of
valuable drops of reagent.



a separation technique in which an electrical
field causes charged molecules to move
through a matrix (usually a gel).
routinely
y used to separate
p
DNA,, p
protein and
other polymeric molecules.
Separation can be based on

Use pause button for momentary spin


The load in a laboratory centrifuge
must be carefully balanced.
Dr. Wenju Lin, BIOL2281_Spring 2016
10

11
Sizes (DNA fragments separated by size).
Net charges
shapes
12
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BIOL2281 E5
The Equipments
Electrophoresis Equipments & Setup

The tray


The comb:


The actual mold which provides a shape for the gel .
It is placed into slots in the tray, with the "teeth" down, when the
agarose is still hot. The agarose polymerizes with small "wells“ into
which samples are added.
The tank

Holds the running buffer, the same buffer used to make the gel
is also used as the running buffer.

One power connection lead is positive (red) and one negative,
there will be a strong electrical current flowing through the tank
when the electrodes are immersed. The gel will be completely
submerged as it is run.
1. The flat-bed tank
2. The gel tray
3. The comb
13
Visualizing DNA by
Ethidium Bromide
Agarose gel electrophoresis

Agarose (nontoxic polysaccharides) gel




14
Mixed with gel running buffer
Melted
Poured into a casting tray
DNA ffragments
t are _____ charged,
h
d th
they will
ill be
b drawn
d
toward
t
d the
th
_____ electrode (anode,red). DNA fragments are separated by
_______.

DNA fragments stained by ethidium bromide (EB) are visible using
UV lamp. EB Intercalates into the DNA molecules and is a
carcinogen.

The lengths of the DNA fragments can be determined by comparing
the migration distances (mm) to the standard curve (log bp vs.
migration distance) of the DNA ladder.
15
 1 g/ml of EB included in
the agarose gel or TBE
buffer
16
Effect of DNA Conformation on
Mobility
500bp DNA ladder

Contains 16 bands in 500bp
increments; the smallest
one is 500 bp

Used to estimate DNA
fragment sizes on a 0.8% 1.0% agarose gel.
wells

The size of uncut plasmid DNA can not be accurately
determined. Uncut plasmid DNA has several distinct
conformations.

Circular
Ci
l fform vs lilinear DNA
Supercoiled vs nicked circles;

1000 bp

The mobility of linear DNA
fragments is inversely
proportional to the log10 of
their molecular weight.
500 bp
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Dr. Wenju Lin, BIOL2281_Spring 2016
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BIOL2281 E5
Agarose gel electrophoresis (con.)

Use of Restriction mapping
The total size of the plasmid can be
obtained by adding up the size of
each DNA band in one lane

A description (roadmap) of restriction
endonuclease cut sites within a region of DNA

To characterize an unknown DNA prior
subcloning or further manipulations, ex:
recombinant plasmid mapping

DNA fingerprinting, ex: restriction digestions on
PCR products, RFLP
Partial digests

Intensity of DNA bands correlate to
the amount or the molecular numbers
of DNA
19
Single digests and double
digests
Plasmid Mapping

Cutting a plasmid
converts a circular
molecule into a linear one.

20
Cutting a plasmid with
two enzymes results in
two linear fragments.

Single digests are used to determine which
restriction sites are in the unknown DNA or to
determine the size of a plasmid.

Double digests are used to order and orient the
fragments correctly.
Enzyme 1
Enzyme 2
How about cutting a linear PCR fragment?
21
22
Draw what you would see on a gel
with the predicted fragments
Predict fragment size based on
plasmid map
Single digest

0
EcoR I
396


EcoRI- 2686bp
Bsey I
Ava II
Negative
electrode
Ava II
2059
pUC 19
Ava II
1837
2,686 bp
Bsey I
1110
Double digest
 EcoRI + Bsey I
1110-396=714,
2686-714=1972
 EcoRI + Ava II
 Bsey I + Ava II
EcoR I+Bsey I
714
2686
DNA Marker
well
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Dr. Wenju Lin, BIOL2281_Spring 2016
1972
EcoR I
500 bp
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BIOL2281 E5
The map of pGLO
386
5371
3181

2106
The restriction site for EcoRV is at 386 and the sites for PstI are at 2106
25
and 3181)
The concentration of an agarose gel allows
for the separation of different sizes of DNA
fragments.
What factors influence the rate of
migration in DNA agarose gel?





26
Molecular size of the DNA
The conformation of DNA
The agarose concentration
The buffer
The applied voltage

0.5% gel : providing better separation for fragments
larger than 10 kb

1% g
gel: providing better separation for fragments
between 500bp-10kb

2% gel: providing better separation for fragments
smaller than 1 kb

0.8% gel will be used in the lab
27
Loading Dye
Voltage

Used to monitor the movement of DNA
fragments, it does not stain DNA
The higher the voltage, the faster the rate of
migration. However,


28
Accompanying heat may melt low-percentage gel.
(Don’tt use more than 130 volts)
(Don
volts).
Imperfections in the gel distort the bands and
produce ambiguous results (slants and smiles).
TBE loading dye contains
 Glycerol
 Bromophenol blue: migrates as a 300bp fragment.
 Xylene cyanol: migrates as a 9000bp (9Kb) fragment.


29
Dr. Wenju Lin, BIOL2281_Spring 2016
10X stock routinely added to the samples before
loading
In the lab: 15µl of reaction mix+ 5µl of 4X loading dye
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BIOL2281 E5
Restriction fragments cut with two
enzymes with complementary tails
In the lab





Complete restriction enzyme digests of pGLO
Set up agarose electrophoresis
Complete Question1 in report E5
Photo of gels will be posted on eLearning
Analyze the plasmid further at home by
visiting the New England Biolabs website and
NCBI website
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Dr. Wenju Lin, BIOL2281_Spring 2016
Sal I
G* TCGA C
C AGCT *G
GG
XHO I
C* TCGA G
G AGCT *C
C
C
CAGCT
CAGCT
GAGCT
GAGCT
TCGAC
TCGAC
+
GG
+
TCGAG
TCGAG
CC
32
6