Mad Cow Dix Agarose Lab

DETECTION
DETECTION OF
OF MAD
MAD COW
COW DISEASE
DISEASE EDVO-Kit
EDVO-Kit 117
117
Background Information
Bovine Spongiform Encephalopathy (BSE), better known as Mad Cow disease, is a fatal condition characterized
by vacuoles (empty spaces) which form within the cytoplasm of neurons. These vacuoles result in a sponge-like
appearance of the brain of the infected animal. Epidemics of BSE has led to the slaughter of tens of thousands of
cattle.
Strong evidence exists that transmission of BSE does not involve nucleic acid vectors (as in a virus) but a protein-
aceous particle known as a prion. The prion is an altered form of an endogenous membrane protein and appears
to self-replicate, following infection, by distorting the shape of its native counterpart (Figure 1). BSE appears
initially in tonsils and other lymphoid organs and then spreads to the nervous system, where it causes apoptosis
(programmed cell death) of neurons. Infected cows become lethargic and later exhibit erratic behavior (hence
the origin of “mad cow disease”).
In addition to cattle, prions also cause neurodegenerative disease in sheep, deer, mink, and humans. A rare,
devastating human disorder known as Creutzfield-Jacob disease (CJD) typically strikes humans over the age of 45;
patients are almost never under 30. In 1996, however, several cases appeared in the United Kingdom in patients
under the age of 25. The brain pathology of these patients was different from classical CJD; this disease was
termed new variant CJD, or "nvCJD". Analysis
revealed that the prion infectious agent from
these patients was identical to the prion from Normal Rogue
BSE-infected cows, suggesting that the pa-
tients became infected by eating contaminat-
ed beef. Other studies suggested that prion
infections could indeed cross species barriers
by expressing the sheep prion gene in mice,
which resulted in mouse spongiform encepha-
lopathy. One theory of the origin of BSE in the
U.K. is that cattle feed may have contained
ruminants of infected sheep, resulting in spe-
cies crossover of the sheep prion to cattle.
Additionally, BSE-infected cow carcasses were
believed to be used to prepare feed, resulting
in widespread propagation of the disease.
Rogue Rogue
Human deaths from nvCJD have been reported
in the UK and France which are believed to
be due to species crossover of BSE to humans.
Widespread panic has devastated the beef
industry in Europe and caused fear of a BSE
outbreak in the United States. One method of
preventing domestic cattle infection is by pro-
hibiting the use of cow or sheep parts in cattle
feed; the U.S. Food and Drug Administration
(FDA) banned this practice in 1997.
To enforce this ban, inspectors test for the
presence of bovine protein in cattle feed. Figure 1:
One method of testing uses the polymerase Conversion of normal protein to distorted form by "Rogue" prion.
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4
EDVO-Kit 117 DETECTION OF MAD COW DISEASE
chain reaction (PCR) on feedstuffs. PCR is a powerful technique universally used to amplify DNA at very specific
sequences. PCR uses a heat-stable enzyme known as Taq DNA polymerase. The reaction mixture contains the
polymerase and two synthetic oligonucleotides, known as “primers” which flank the sequence(s) to be ampli-
fied, known as the “template”. In the case of cattle feed compliance, the template would be feedstuffs from
various feed mills and the primers would be complimentary to a bovine-specific gene.
In the first step of a PCR reaction (Figure 2, next page), the template complimentary DNA strands are melted/
separated from each other at 94°C, at which temperature the Taq DNA polymerase remains stable. In the second
step, known as annealing, the sample is cooled to allow hybridization of the primers to the two strands of the
target sequence(s). In the third step, known as extension, the temperature is raised to 72°C and the Taq poly-
merase adds nucleotides to the primers to complete synthesis of the new complementary strands. The three
steps - denaturation, an-
nealing, and extension -
constitute one PCR “cycle”. T arget Sequence
This process is typically 5' 3'
repeated for 20-30 cycles, 3' 5'
= Separation of
amplifying the target two DNA strands
sequence exponentially = Primer 1
(Figure 2, bottom). PCR is
= Primer 2 5' 3'
performed in a thermal cy- Denature
cler, which is programmed 94°C
to rapidly heat, cool and 3' 5'
Cycle 1
maintain samples at des-

ignated temperatures for
varying amounts of time. 5' 3'
5'
In this experiment, a Anneal
5' 2 primers
hypothetical scenario 3' 5' 40°C - 65°C
involves the U.S. Fed-
eral Drug Administration
5' 5' 3' Extension
(FDA) laboratory, which 3' 5' 5' 72°C
has obtained cattle feed
samples from different
feed mills. Using bovine
specific primers, PCR has
been performed on each of 5' 5' 3'
Cycle 2
these samples. Students 3' 5' 5'

submit the samples to 5' 5' 3'
agarose gel electrophoresis 3' 5' 5'
to determine if any of the
cattle feed samples contain
bovine proteins, which
could propagate mad cow 5' 5' 3'
disease. The presence 3' 5' 5'
of an amplified product
Cycle 3
5' 3' 5' 3'

indicates the presence of 3' 5' 5'
bovine products in the 5' 5' 3'
cattle feed, in violation of 3' 5' 3' 5'
the federal statute. 5' 5' 3'
3' 5' 5'
Figure 2: DNA Amplification by the Polymerase Chain Reaction
5
DETECTION
DETECTION OF
OF MAD
MAD COW
COW DISEASE
DISEASE EDVO-Kit
EDVO-Kit 117
117
Experiment Overview
EXPERIMENT OBJECTIVE:
The objective of this experiment is to educate students about Bovine Spongiform Encephalopathy (BSE), better
known as Mad Cow disease.
LABORATORY SAFETY
1. Gloves and goggles should be worn routinely as good laboratory practice.
2. Exercise extreme caution when working with equipment that is used in conjunction
with the heating and/or melting of reagents.
Wear gloves
and safety goggles
3. DO NOT MOUTH PIPET REAGENTS - USE PIPET PUMPS.
4. Exercise caution when using any electrical equipment in the laboratory.

5. Always wash hands thoroughly with soap and water after handling reagents or biological materials in the
laboratory.
LABORATORY NOTEBOOKS:
Scientists document everything that happens during an experiment, including experimental conditions,
thoughts and observations while conducting the experiment, and, of course, any data collected. Today, you’ll
be documenting your experiment in a laboratory notebook or on a separate worksheet.
Before starting the Experiment:
• Carefully read the introduction and the protocol. Use this information to form a hypothesis for this
experiment.
• Predict the results of your experiment.
During the Experiment:
• Record your observations.
After the Experiment:
• Interpret the results – does your data support or contradict your hypothesis?
• If you repeated this experiment, what would you change? Revise your hypothesis to reflect this
change.
6
Experiment Overview
Prepare
1 agarose gel in
casting tray
2 Remove end
blocks & comb,
then submerge
gel under buffer in
electrophoresis
chamber
3 Load each
sample in
consecutive wells
4 Attach safety
cover,connect
leads to power
source and conduct
electrophoresis
5 After electrophoresis,
transfer gel for staining
(-)
InstaStain® Blue
1 2 3 4 5 6
or FlashBlue™
DNA stain.
Analysis on
white light
source.
(+)
Gel pattern will vary

depending upon experiment.
7
DETECTION
DETECTION OF
OF MAD
MAD COW
COW DISEASE
DISEASE EDVO-Kit
EDVO-Kit 117
117
Module I: Agarose Gel Electrophoresis
1. 50
2. 3. 1:00
x IMPORTANT:
Concentrated Distilled If you are unfamiliar with
Agarose
buffer water agarose gel prep and
electrophoresis, detailed
Flask instructions and helpful
Caution! Flask will be HOT! resources are available at
www.edvotek.com
4. 5.
60°C
Wear gloves
and safety goggles
6. WAIT 7.
Pour
20
min.
60°C
© 2013 Edvotek® All Rights Reserved.
CASTING THE AGAROSE GEL

1. DILUTE concentrated (50X) buffer with distilled water to create 1X buffer (see Table A).
1. MIX
2. DILUTEagarose powder with 1X
concentrated 50X buffer in a 250 ml flask (see
Electrophoresis Tablewith
buffer A). distilled water (refer to Table A for correct volumes
3. DISSOLVE
depending on the size of your gel casting tray). the solution on high for 1 minute. Carefully REMOVE the flask from
agarose powder by boiling the solution. MICROWAVE
the
2. MIX microwave
agarose MIX by swirling
andpowder with bufferthe flask.solution to HEAT
Continue in a 250 theml
solution
flaskin(refer
15-second bursts until
to Table A). the agarose is completely dissolved
3. (the solution should
DISSOLVE agarose be clear like water).
powder by boiling the solution. MICROWAVE the solution on high for 1 minute. Care-
4. COOL
fully agarose
REMOVE to 60°
theC flask
with careful
fromswirling to promote even
the microwave anddissipation
MIX by ofswirling
heat. the flask. Continue to HEAT the solution in
5. While agarose bursts
15-second is cooling, SEALthe
until the agarose
ends of theisgel-casting tray with
completely the rubber(the
dissolved end caps. PLACEshould
solution the wellbe
template
clear (comb) in the
like water).
COOL agarose
4. appropriate notch. to 60° C with careful swirling to promote even dissipation of heat.
5. Whiletheagarose
6. POUR cooled agarose solutionSEAL
is cooling, into thethe ends gel-casting
prepared of the gel-casting
tray. The gel tray
shouldwith the rubber
thoroughly end caps.
solidify within PLACE
20 minutes. The the well
gel will
template
stiffen (comb)
and become lessin the appropriate
transparent notch.
as it solidifies.
6. POUR the
7. REMOVE end cooled
caps and agarose
comb. Takesolution intowhen
particular care the removing
pre- the combTable to prevent damage to the wells.
pared gel-casting tray. The gel should thoroughly
solidify within 20 minutes. The gel will stiffen and
A Individual 0.8% UltraSpec-Agarose™ Gel
Table Size of Gel Concentrated Distilled Amt of TOTAL

Abecome Individual 0.8% UltraSpec-Agarose™
less transparent as it solidifies.Gel Casting tray Buffer (50x) + Water + Agarose = Volume
7. REMOVE end caps and comb. Take particular care
7 x 7 cm 0.6 ml 29.4 ml 0.23 g 30 ml
Size of Gel
when removing the comb
Concentrated Distilled toAmt
prevent
of
Casting tray Buffer (50x) + Water + Agarose =
damage
TOTAL
Volume
to
the wells. 7 x 10 cm 1.0 ml 49.0 ml 0.39 g 50 ml
7 x 7 cm 0.6 ml 29.4 ml 0.23 g 30 ml
7 x 14 cm 1.2 ml 58.8 ml 0.46 g 60 ml
7 x 10 cm 1.0 ml 49.0 ml 0.39 g 50 ml
7 x 14 cm 1.2 ml 58.8 ml 0.46 g 60 ml
8
Module I: Agarose Gel Electrophoresis
8. 9.
Pour
1X Diluted
Buffer
10. 11.
Wear gloves
and safety goggles
REMINDER:
Before loading the
samples, make sure
the gel is properly
oriented in the ap-
© 2013 Edvotek® All Rights Reserved. paratus chamber.
12. 1 2
(-)
3 4 8. PLACE gel (on the tray) into electrophoresis chamber. Completely COVER the gel
5 6
RUNNING THE GEL with 1X electrophoresis buffer (See Table B for recommended volumes).
9. LOAD 25 µl of the DNA samples into wells in consecutive order.
8. PLACE the gel (still on the 10. PLACE
tray) safetythe
into CHECK that the gel is properly
cover.electrophoresis oriented. COVER
chamber. Remember, the
the DNAgel with 1X Electrophoresis
samples will migrate toward the positive (red) electrode.
Buffer (See Table B for recommended volumes). The gel should be completely
11. CONNECT leads to the power source and PERFORM electrophoresis (See Table C
submerged.
9. PUNCTURE (the
+)
foil overlay offorthe
timeQuickStrip™ with a pipet tip. LOAD the entire sample (35 µl) into the
and voltage guidelines).
well in the order indicated12.byAfter
Table 1, at right.
electrophoresis is complete, REMOVE the gel and casting tray from the
10. PLACE safety cover on the unit. CHECKchamber
electrophoresis that and theproceed
gel toisSTAINING
properly & VISUALIZING the results.
oriented. Remember, the DNA samples will migrate toward the

Table 1: Gel Loading
positive (red) electrode.
11. CONNECT leads to the power source and PERFORM electropho- Lane 1 Tube A Standard DNA Marker
resis (See Table C for time and voltage guidelines). Allow the 2 Tube B Positive bovine protein control
tracking dye to migrate at least 3.5 cm from the wells. 3 Tube C Negative bovine protein control
12. After electrophoresis is complete, REMOVE the gel and casting
4 Tube D Feed sample from mill #1
tray from the electrophoresis chamber and proceed to instruc-
5 Tube E Feed sample from mill #2
tions for STAINING the agarose gel.
6 Tube F Feed sample from mill #3
Table Table
B 1x Electrophoresis Buffer (Chamber Buffer)

C Time & Voltage Guidelines (0.8% Agarose Gel)
Electrophoresis Model
Dilution
EDVOTEK Total Volume M12 (classic)
50x Conc. Distilled M6+ M12 (new)
Model # Required
Buffer + Water
& M36
Volts Min. / Max. Min. / Max. Min. / Max.
M6+ & M12 (new) 300 ml 6 ml 294 ml 150 15/20 min. 20/30 min. 25 / 35 min.
M12 (classic) 400 ml 8 ml 392 ml 125 20/30 min. 30/35 min. 35 / 45 min.
M36 1000 ml 20 ml 980 ml 75 35 / 45 min. 55/70 min. 60 / 90 min.
9
DETECTION
DETECTION OF
OF MAD
MAD COW
COW DISEASE
DISEASE EDVO-Kit
EDVO-Kit 117
117
Module II-A: Staining Agarose Gels Using FlashBlue™
1. 10
2.
x
Concentrated Distilled
FlashBlue™ Stain water
Wear gloves
Flask and safety goggles
3. 4. 5. 1 2
(-)
3 4 5 6
DESTAIN
STAIN
Pour
Pour
5 20
min.
min.
(+)
1. DILUTE 10 ml of 10x concentrated FlashBlue™ with 90 ml of water in a flask and MIX well.
2. REMOVE the agarose gel and casting tray from the electrophoresis chamber. SLIDE the gel off of the cast-
ing tray into a small, clean gel-staining tray.
3. COVER the gel with the 1x FlashBlue™ stain solution. STAIN the gel for 5 minutes. For best results, use an
orbital shaker to gently agitate the gel while staining. STAINING THE GEL FOR LONGER THAN 5 MINUTES
WILL REQUIRE EXTRA DESTAINING TIME.
4. TRANSFER the gel to a second small tray. COVER the gel with water. DESTAIN for at least 20 minutes
with gentle shaking (longer periods will yield better results). Frequent changes of the water will acceler-
ate destaining.
5. Carefully REMOVE the gel from the destaining liquid. VISUALIZE results using a white light visualization
system. DNA will appear as dark blue bands on a light blue background.
ALTERNATIVE PROTOCOL:
1. DILUTE one ml of concentrated FlashBlue™ stain with 149 ml dH2O.

2. COVER the gel with diluted FlashBlue™ stain.
3. SOAK the gel in the staining liquid for at least three hours. For best results, stain gels overnight.
10
EDVO-Kit 117
EDVO-Kit 117 DETECTION OF
DETECTION OF MAD
MAD COW
COW DISEASE
DISEASE
Module II-B: Staining Agarose Gels Using InstaStain® Blue
1. 2. 3. 4.
Moisten tain
® Blu
e
the gel
taS
Ins in g
d
Pen
ent
. Pat
U.S
® Ethid
InstaStain
t Pending
U.S. Paten
5. 6. 7. 8. (-)
- 75 ml
-
- STAIN
-
DESTAIN
10
- Blu
e
®
tain
taS
Ins ing
20
d
Pen
ent
min.
. Pat
U.S
InstaStain® Ethidium
Bromide
min.
Pending
U.S. Patent
or overnight
(+)
1. Carefully REMOVE the agarose gel and casting tray from the electrophoresis chamber.
SLIDE the gel off of the casting tray on to a piece of plastic wrap on a flat surface.
2. MOISTEN the gel with a few drops of electrophoresis buffer.Cover & Soak
3. 1.Wearing gloves,75 ml 2. blue side of the InstaStain®
PLACE the 3. 3 hourscard
Blue 4. gel.
or on the (-)
overnight
4. With a gloved hand, REMOVE air bubbles between the card and the gel by firmly run-
Ins
taS
tain
® Blu
Wear gloves
e
ning your fingers over the entire surface. Otherwise, those regions will not stain. and safety goggles
5. PLACE the casting tray on top of the gel/card stack. PLACE a small weight (i.e. an
empty glass beaker) on top of the casting tray. This ensures that the InstaStain® Blue NOTE:
(+)
card is in direct contact with the gel surface. STAIN the gel for 10 minutes. DO NOT STAIN
6. REMOVE the InstaStain® Blue card. If the color of the gel appears very light, reapply GELS IN THE
the InstaStain® Blue card to the gel for an additional five minutes. ELECTROPHORESIS
7. TRANSFER the gel to a small, clean gel-staining tray. COVER the gel with about 75 mL APPARATUS.
of distilled water and DESTAIN for at least 20 minutes. For best results, use an orbital
shaker to gently agitate the gel while staining. To accelerate destaining, warm the
distilled water to 37°C and change it frequently.
8. Carefully REMOVE the gel from the destaining liquid. VISUALIZE results using a white light
visualization system. DNA will appear as dark blue bands on a light blue background.
ALTERNATIVE PROTOCOL:
1. Carefully SLIDE the agarose gel from its casting tray into a small, clean tray containing about 75 ml of dis-
tilled/deionized water or used electrophoresis buffer. The gel should be completely submerged.
2. Gently FLOAT the InstaStain® Blue card(s) on top of the liquid with the stain (blue side) facing toward the gel.
Each InstaStain® Blue card will stain 49 cm2 of gel (7 x 7 cm).
3. COVER the tray with plastic wrap to prevent evaporation. SOAK the gel in the staining liquid for at least 3
hours. The gel can remain in the liquid overnight if necessary.
4. Carefully REMOVE the gel from the staining tray. VISUALIZE results using a white light visualization system.
DNA will appear as dark blue bands on a light blue background.
11
DETECTION
DETECTION OF
OF MAD
MAD COW
COW DISEASE
DISEASE EDVO-Kit
EDVO-Kit 117
117
Study Questions
1. What is a prion? How might a prion-based disease be transmitted?
2. What is bovine spongiform encephalopathy? What are some characteristics of this condition?
3. What is Creutzfield-Jacob disease? How is it contracted?
12

Mad Cow Dix Agarose Lab

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DETECTION

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maintain samples at des-

these samples. Students 3' 5' 5'

5' 3' 5' 3'

Figure 2: DNA Amplification by the Polymerase Chain Reaction

1.800.EDVOTEK • Fax 202.370.1501 • info@edvotek.com • www.edvotek.com

1. Gloves and goggles should be worn routinely as good laboratory practice.

4. Exercise caution when using any electrical equipment in the laboratory.

Before starting the Experiment:

During the Experiment:

• Record your observations.

After the Experiment:

1.800.EDVOTEK • Fax 202.370.1501 • info@edvotek.com • www.edvotek.com

Gel pattern will vary

1.800.EDVOTEK • Fax 202.370.1501 • info@edvotek.com • www.edvotek.com

Module I: Agarose Gel Electrophoresis

© 2013 Edvotek® All Rights Reserved.

CASTING THE AGAROSE GEL

Table Size of Gel Concentrated Distilled Amt of TOTAL

7 x 14 cm 1.2 ml 58.8 ml 0.46 g 60 ml

1.800.EDVOTEK • Fax 202.370.1501 • info@edvotek.com • www.edvotek.com

Module I: Agarose Gel Electrophoresis

oriented. Remember, the DNA samples will migrate toward the

B 1x Electrophoresis Buffer (Chamber Buffer)

1.800.EDVOTEK • Fax 202.370.1501 • info@edvotek.com • www.edvotek.com

Module II-A: Staining Agarose Gels Using FlashBlue™

1. DILUTE one ml of concentrated FlashBlue™ stain with 149 ml dH2O.

1.800.EDVOTEK • Fax 202.370.1501 • info@edvotek.com • www.edvotek.com

Module II-B: Staining Agarose Gels Using InstaStain® Blue

1.800.EDVOTEK • Fax 202.370.1501 • info@edvotek.com • www.edvotek.com

1. What is a prion? How might a prion-based disease be transmitted?

3. What is Creutzfield-Jacob disease? How is it contracted?

1.800.EDVOTEK • Fax 202.370.1501 • info@edvotek.com • www.edvotek.com

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