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Edvo-Kit #335
Reverse Transcription PCR (RT-PCR):
The Molecular Biology of HIV Replication
Experiment Objective:
The objective of this experiment is for students to gain an understanding
of the principles and practice of RT-PCR and to relate these reactions to
HIV replication.
335.151030
Reverse Transcription PCR (RT-PCR): The Molecular Biology of HIV Replication EDVO-Kit
EDVO-Kit 335
335
Table of Contents
Page
Experiment Components 3
Experiment Requirements 4
Background Information 5
Experiment Procedures
Experiment Overview 9
Module I: RT-PCR Reaction 10
Module II: Agarose Gel Electrophoresis 11
Module III: Staining Agarose Gels with InstaStain Ethidium Bromide 13
Module IV: Size Determination of RT-PCR Amplied Fragment 14
Study Questions 17
Instructor's Guidelines 18
Pre-Lab Preparations 19
Experiment Results and Analysis 23
Study Questions and Answers 24
Appendices 25
A EDVOTEK Troubleshooting Guide 26
B Preparation and Handling of PCR Samples With Wax 27
C Bulk Preparation of Agarose Gels 28
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EDVO-Kit 335
EDVO-Kit 335 Reverse Transcription PCR (RT-PCR): The Molecular Biology of HIV Replication
Experiment Components
CAUTION!
Wear gloves when handling all tubes for this experiment.
RNA from your fingers will interfere with the experiment results.
EDVOTEK, The Biotechnology Education Company, and InstaStain are registered trademarks of EDVOTEK, Inc.
EdvoBead, UltraSpec-Agarose and EdvoQuick are trademarks of EDVOTEK, Inc.
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Reverse Transcription PCR (RT-PCR): The Molecular Biology of HIV Replication EDVO-Kit
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Requirements
*If you do not have a thermal cycler, PCR experiments can be conducted, with proper care, using three waterbaths.
However, a thermal cycler assures a signicantly higher rate of success.
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EDVO-Kit 335
EDVO-Kit 335 Reverse Transcription PCR (RT-PCR): The Molecular Biology of HIV Replication
Background Information
Acquired immune deciency syndrome (AIDS) is a disease characterized by the progressive deterioration of a
patient's immune system. This immunological impairment allows infectious agents such as viruses, bacteria, fungi
and parasites to invade the body and propagate. The incidence of certain cancers dramatically increases in these
patients because of their compromised immune system. AIDS is a serious threat to human health and is a global
problem. Intensive research is
being done to advance meth-
ods of detection, clinical treat- Protease
ment and prevention. Reverse
Glycoprotein transcriptase
Retroviruses gp 120 Protein
gp 160 coat
The AIDS etiologic agent is the gp 41
human immunodeciency virus
type 1 (HIV-1), a retrovirus. RNA
Retroviruses contain an RNA ge-
nome and the RNA-dependent
DNA polymerase also termed
reverse transcriptase. Members Core
of the retrovirus family are Lipid
involved in the pathogenesis bilayer
of certain types of leukemias Integrase
and other sarcomas in animals.
The structure and replication
mechanism of HIV is very simi-
lar to other retroviruses. HIV is Figure 1: Overview of HIV structural components
unique in some of its properties
since it specically targets the
immune system, is very immu-
noevasive, forms signicant amounts of progeny virus in vivo during the later stages of the disease and can also be
transmitted during sexual activity.
The HIV viral particle is surrounded by a lipid bilayer derived from the host cell membrane during budding (Figure
1). The viral proteins are identied by the prex gp (glycoprotein) or p (protein) followed by a number indicating
the approximate molecular weight in kilodaltons. The lipid bilayer is studded with a protein complex called gp160,
a protein complex made from gp120 and gp41. The gp 41 anchors gp 120 in the bilayer.
Beneath the bilayer is a capsid consisting of p17 and p18. Within this shell is the viral core. The walls of the core
consists of p24 and p25. Within the core are two identical RNA molecules 9000 nucleotides in length. Hydrogen
bonded to each genomic RNA is a cellular tRNA molecule. The core also contains reverse transcriptase. Protein
products obtained from the HIV genome are displayed in Figure 2.
Large quantities of virus can be grown in tissue culture for diagnostic and research purposes. Several of the viral
proteins have been cloned and expressed in relatively large quantities.
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Reverse Transcription PCR (RT-PCR): The Molecular Biology of HIV Replication EDVO-Kit 335
Biology of HIV
GAG/POL Infectivity and Replication
Infection overlap Replication Genes Gene
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EDVO-Kit 335 Reverse Transcription PCR (RT-PCR): The Molecular Biology of HIV Replication
RNA
HIV Core
IMMUNE
HIV
CELL
Cell cytoplasm
Reverse
Transcriptase
Newly made
viral RNA
New virus
budding out
of the cell
New HIV
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Reverse Transcription PCR (RT-PCR): The Molecular Biology of HIV Replication EDVO-Kit 335
see why treatments with a single drug have failed. HIV is different from other viruses that mutate rapidly because
it stays in the body for years, replicating again and again. This large number of rounds of replication multiplied by
the high mutation rate makes HIV uniquely dangerous to the host.
Researchers continue to examine each stage of the life cycle as well as to note the various ways the immune sys-
tem ghts HIV at different stages. Many drugs called antiretrovirals that ght HIV infection are based on differ-
ent aspects of the structure and function of the HIV molecule.
Through a complex mechanism involving several events, reverse transcriptase synthesizes a double stranded DNA
copy of the genomic RNA template. Transfer RNA acts as the primer for the rst DNA strand synthesis, resulting
in an RNA-DNA hybrid. RNAse H degrades the RNA strand of the RNA-DNA duplex and the polymerase activity
synthesizes a complementary DNA strand.
Unlike other cellular DNA polymerases, HIV DNA polymerase (reverse transcriptase) has a high error rate (1 in
104). The frequent mutations change the viral protein epitopes. This is believed to be the main mechanism of HIV
immunoevasion. The double stranded DNA (dsDNA) migrate into the cell nucleus where they become covalently
integrated into the cellular genomic DNA. This integration is catalyzed by HIV integrase.
The viral DNA integrates via specic, self-complimentary sequences at both ends called long terminal repeats,
or LTRs (see Figure 2). The integrated DNA is called proviral DNA or the provirus. The provirus enters a period of
latency that can last for several years. The proviral DNA is replicated along with the cellular DNA and can be inher-
ited through many generations.
The HIV proviral DNA contains the major genes common to all non-transducing retroviruses. These genes are gag,
pol and env (see Figure 2). HIV also contains ve or six other genes that are much smaller. Retroviral transcription
is a complex process producing a variety of RNAs. Production of transcripts is controlled in the LTR and transcrip-
tional termination signals are located in each major gene. The RNA transcripts that remain unspliced become
packaged in the new viral particles.
The gag gene is translated into a polypeptide that is cleaved by a viral protease into four proteins that form the
inner shells. Specic protease inhibitors are clinically being used to inhibit protein processing and control the
further spread of the HIV virus in patients suffering from AIDS. The pol gene encodes the reverse transcriptase and
the integrase which is responsible for the genomic incorporation of copy DNA. The env gene encodes the surface
glycoproteins the viral particles acquire as they bud from the cells.
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EDVO-Kit 335
EDVO-Kit 335 Reverse Transcription PCR (RT-PCR): The Molecular Biology of HIV Replication
Experiment Overview
EXPERIMENT OBJECTIVE:
The objective of this experiment is for students to gain an understanding of the principles
and practice of RT-PCR and to relate these reactions to HIV replication.
Be sure to READ and UNDERSTAND the instructions completely BEFORE starting the
experiment. If you are unsure of something, ASK YOUR INSTRUCTOR!
Wear gloves and goggles while working in the laboratory.
Exercise caution when working in the laboratory you will be using equipment that can be dangerous if
used incorrectly.
Wear protective gloves when working with hot reagents like boiling water and melted agarose.
DO NOT MOUTH PIPET REAGENTS - USE PIPET PUMPS.
Always wash hands thoroughly with soap and water after working 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 docu-
menting your experiment in a laboratory notebook or on a separate worksheet.
Carefully read the introduction and the protocol. Use this information to form a hypothesis for this ex-
periment.
Predict the results of your 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 reect this change.
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Reverse Transcription PCR (RT-PCR): The Molecular Biology of HIV Replication EDVO-Kit
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335
1. 2. 3.
RT-PCR
CONTROL
RT-PCR
RT-PCR
CONTROL
10 l primer mix
10 l RNA
30 l RNase-free
water
RT-PCR
RT-PCR
CONTROL
CONTROL
1. LABEL two PCR tubes with RT-PCR or Control plus your group name or number.
2. To each tube, ADD 10 l RT-PCR primer mix, 10 l RNA, and 30 l RNase-free water.
3. ADD the RT-PCR EdvoBead to the tube labeled RT-PCR.
4. MIX the samples gently. Make sure the RT-PCR EdvoBead is completely dissolved.
5. CENTRIFUGE to collect the sample at the bottom of the tube.
6. AMPLIFY sample using RT-PCR:
Reverse Transcription:
65C for 40 minutes
PCR cycling conditions:
Denaturation 94C for 5 minutes
94C for 30 seconds
50C for 30 seconds 35 cycles
72C for 30 seconds
Final Extension 72C for 5 minutes
7. After RT-PCR, ADD 5 l 10X Gel Loading Dye to the samples. PLACE tubes on ice. PROCEED to
Module II: Agarose Gel Electrophoresis.
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EDVO-Kit 335
EDVO-Kit 335 Reverse Transcription PCR (RT-PCR): The Molecular Biology of HIV Replication
1. 50
2. 3. 1:00 IMPORTANT:
x
7 x 14 cm gels are
Concentrated Distilled Agarose recommended. Each gel
buffer water can be shared by two
Flask
groups. Place well-former
template (comb) in the
Caution! Flask will be HOT!
rst set of notches.
If you are unfamiliar with
4. 5. agarose gel prep and
electrophoresis, detailed
instructions and helpful
resources are available at
60C www.edvotek.com
6. WAIT 7.
Pour
20
min.
60C Wear gloves
and safety goggles
1. DILUTE concentrated (50X) buffer with distilled water to create 1X buffer (see Table A).
2. MIX agarose powder with 1X buffer in a 250 ml ask (see Table A).
3. DISSOLVE agarose powder by boiling the solution. MICROWAVE the solution on high for 1 minute. Care-
fully REMOVE the ask from the microwave and MIX by swirling the ask. Continue to HEAT the solution in
15-second bursts until the agarose is completely dissolved (the solution should be clear like water).
4. COOL agarose to 60 C with careful swirling to promote even dissipation of heat.
5. While agarose is cooling, SEAL the ends of the gel-casting tray with the rubber end caps. PLACE the well
template (comb) in the appropriate notch.
6. POUR the cooled agarose solution into the prepared gel-casting tray. The gel should thoroughly solidify
within 20 minutes. The gel will stiffen and become less
transparent as it solidies.
Table
7. REMOVE end caps and comb. Take particular care when
removing the comb to prevent damage to the wells.
A Individual 1.0% UltraSpec-Agarose Gel
Size of Gel Concentrated Distilled Amt of TOTAL
Casting tray Buffer (50x) + Water + Agarose = Volume
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Reverse Transcription PCR (RT-PCR): The Molecular Biology of HIV Replication EDVO-Kit 335
8. Pour 9.
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-
paratus chamber.
8. PLACE gel (on the tray) into electrophoresis chamber. COVER the gel
with 1X electrophoresis buffer (See Table B for recommended vol-
Table 1
umes). The gel should be completely submerged.
9. LOAD 25 l into the well in the order indicated by Table 1. Lane Recommended
10. PLACE safety cover. CHECK that the gel is properly oriented. Remem-
1 EdvoQuick DNA Ladder
ber, the DNA samples will migrate toward the positive (red) electrode.
2 Control Sample Group 1
11. CONNECT leads to the power source and PERFORM electrophoresis
(See Table C for time and voltage guidelines). 3 RT-PCR Sample Group 1
12. After electrophoresis is complete, REMOVE the gel and casting tray 4 Control Sample Group 2
from the electrophoresis chamber and proceed to STAINING the aga- 5 RT-PCR Sample Group 2
rose gel.
Table
1x Electrophoresis Buffer (Chamber Buffer)
B Table
Time and Voltage Guidelines
EDVOTEK Total Volume
Dilution C (1.0% - 7 x 14 cm Agarose Gel)
50x Conc. Distilled
Model # Required
Buffer + Water
Recommended Time
Volts Minimum Maximum
M6+ 300 ml 6 ml 294 ml 125 55 min. 1 hour 15 min.
M12 400 ml 8 ml 392 ml 70 2 hours 15 min. 3 hours
M36 1000 ml 20 ml 980 ml 50 3 hours 25 min. 5 hours
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EDVO-Kit 335
EDVO-Kit 335 Reverse Transcription PCR (RT-PCR): The Molecular Biology of HIV Replication
1. 2. 3.
Moisten in
Eth
idiu
m Bro
m id
e
the gel
Sta in g
sta end
In nt P
ate
.P
U.S
4. 5. 6.
-
-
- STAIN 300 nm
-
-
3-5
Ethid
min.
InstaStain
m Bromide
InstaStain Ethidiu
nt Pending
U.S. Pate
Pending
U.S. Patent
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
at surface. DO NOT STAIN GELS IN THE ELECTROPHORESIS APPARATUS.
2. MOISTEN the gel with a few drops of electrophoresis buffer.
3. Wearing gloves, REMOVE and DISCARD the clear plastic protective sheet from Wear gloves and
UV safety goggles
the unprinted side of the InstaStain card(s). PLACE the unprinted side of the
InstaStain Ethidium Bromide card(s) on the gel. You will need 2 cards to stain
a 7 x 14 cm gel.
4. With a gloved hand, REMOVE air bubbles between the card and the gel by rmly
running your ngers over the entire surface. Otherwise, those regions will not
stain.
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 In-
staStain Ethidium Bromide card is in direct contact with the gel surface. STAIN
the gel for 3-5 minutes.
6. REMOVE the InstaStain Ethidium Bromide card(s). VISUALIZE the gel using a
mid-range ultraviolet transilluminator (300 nm). DNA should appear as bright
orange bands on a dark background.
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Reverse Transcription PCR (RT-PCR): The Molecular Biology of HIV Replication EDVO-Kit
EDVO-Kit 335
335
1 2 3
Agarose gel electrophoresis separates DNA into discrete bands, each
comprising molecules of the same size. How can these results be used to
determine the lengths of different fragments? Remember, as the length Figure 5:
of a DNA increases, the distance to which the molecule can migrate de- Measure distance
creases because large molecules cannot pass through the channels in the migrated from the
lower edge of the
gel with ease. Therefore, the migration rate is inversely proportional to
well to the lower
the length of the moleculesmore specically, to the log10 of molecule's edge of each band.
length. To illustrate this, we ran a sample that contains bands of known
lengths called a "standard". We will measure the distance that each of
these bands traveled to create a graph, known as a "standard curve",
which can then be used to extrapolate the size of unknown molecule(s).
Figure 6: Semilog graph example
1. Measure and Record Migration Distances
Measure the distance traveled by each Stan-
dard DNA Fragment from the lower edge of the
sample well to the lower end of each band. Re-
cord the distance in centimeters (to the nearest
millimeter) in your notebook. Repeat this for
each DNA fragment in the standard.
Measure and record the migration distances of
each of the fragments in the unknown samples
in the same way you measured the standard
bands.
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EDVO-Kit 335 Reverse Transcription PCR (RT-PCR): The Molecular Biology of HIV Replication
After all the points have been plotted, use a ruler or a straight edge to draw the best straight line possible
through the points. The line should have approximately equal numbers of points scattered on each side of the
line. It is okay if the line runs through some points (see Figure 6 for an example).
a. Locate the migration distance of the unknown fragment on the x-axis of your semi-log graph. Draw a
vertical line extending from that point until it intersects the line of your standard curve.
b. From the point of intersection, draw a second line, this time horizontally, toward the y-axis. The value at
which this line intersects the y-axis represents the approximate size of the fragment in base pairs (refer
to Figure 6 for an example). Make note of this in your lab notebook.
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Reverse Transcription PCR (RT-PCR): The Molecular Biology of HIV Replication EDVO-Kit 335
10,000
9,000
8,000
7,000
6,000
5,000
4,000
3,000
2,000
1,000
900
800
700
600
500
Y-axis: Base Pairs
400
300
200
100
90
80
70
60
50
40
30
20
10
1 cm 2 cm 3 cm 4 cm 5 cm 6 cm
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EDVO-Kit 335
EDVO-Kit 335 Reverse Transcription PCR (RT-PCR): The Molecular Biology of HIV Replication
Study Questions
3. What is the source of the four dXTPs required for DNA synthesis?
4. What is the source of the tRNA primer and what is its function in the viral replication?
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INSTRUCTOR'S GUIDE Reverse Transcription PCR (RT-PCR): The Molecular Biology of HIV Replication) EDVO-Kit 335
Instructor's Guide
OVERVIEW OF INSTRUCTORS PRELAB PREPARATION:
This section outlines the recommended prelab preparations and approximate time requirement to complete each
prelab activity.
Module III:
Staining Agarose Gels Prepare staining The class period or overnight after the 10 min.
with InstaStain components class period.
Ethidium Bromide
Module IV:
Size Determination of Any time before the class. 5 min.
Print Semi-log Paper
PCR Amplified DNA
Fragment
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EDVO-Kit 335 Reverse Transcription PCR (RT-PCR): The Molecular Biology of HIV Replication) INSTRUCTOR'S GUIDE
2. Add 500 l of RNase-free water (D) to the tube of Primer Mix Concentrate
(B). Cap tubes and mix.
Continued
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INSTRUCTOR'S GUIDE Reverse Transcription PCR (RT-PCR): The Molecular Biology of HIV Replication) EDVO-Kit 335
PREPARATION OF RNA
2. Add 135 l of RNase-free water (D) to the tube containing RNA Template Concentrate (A). Pipet up and
down to mix.
3. Aliquot 25 l of the diluted RNA control into appropriately labeled microcentrifuge tubes. Place the tubes on
ice until they are needed.
ADDITIONAL MATERIALS
Dispense 50 l of 10X Gel Loading Solution per tube. Label these 6 tubes "10x Solution". Distribute one tube
per student group
Dispense 100 l of RNAse-free water (D) per tube. Label these 10 tubes "Water". Distribute one tube per
student group.
Each group will also receive two PCR tubes and one RT-PCR EdvoBead. For best results, use RT-PCR Edvo-
Beads within two weeks of receipt. The RT-PCR EdvoBEads should arrive as small, white, spherical pellets
in a plastic tube. If the bead appears to be shriveled or melted, please contact EDVOTEK customer service
before performing the experiment.
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EDVO-Kit 335 Reverse Transcription PCR (RT-PCR): The Molecular Biology of HIV Replication) INSTRUCTOR'S GUIDE
Additional Materials:
Each 1.0% gel should be loaded with the EdvoQuick DNA ladder and PCR reactions
from two student groups.
FOR MODULE II
Each Group should receive:
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INSTRUCTOR'S GUIDE Reverse Transcription PCR (RT-PCR): The Molecular Biology of HIV Replication) EDVO-Kit 335
Pre-Lab Preparations
InstaStain Ethidium Bromide provides the sensitivity of ethidium bromide while minimiz-
Wear gloves
ing the volume of liquid waste generated by staining and destaining a gel. An agarose and safety goggles
gel stained with InstaStain Ethidium Bromide is ready for visualization in as little as 3
minutes! Each InstaStain card will stain 49 cm2 of gel (7 x 7 cm). You will need 2 cards
to stain a 7 x 14 cm gel.
Use a mid-range ultraviolet transilluminator (Cat. #558) to visualize gels stained with InstaStain
Ethidium Bromide. BE SURE TO WEAR UV-PROTECTIVE EYEWEAR!
Standard DNA markers should be visible after staining even if other DNA samples are faint or ab-
sent. If bands appear faint, repeat staining with a fresh InstaStain card for an additional 3-5 min.
If markers are not visible, troubleshoot for problems with electrophoretic separation.
Ethidium bromide is a listed mutagen. Wear gloves and protective eyewear when using this prod-
uct. UV protective eyewear is required for visualization with a UV transilluminator.
InstaStain Ethidium Bromide cards and stained gels should be discarded using institutional guide-
lines for solid chemical waste.
Once gels are stained, you may wish to photograph your results. There are many different photod-
ocumentation systems available, including digital systems that are interfaced directly with computers.
Specic instructions will vary depending upon the type of photodocumentation system you are using.
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EDVO-Kit 335 Reverse Transcription PCR (RT-PCR): The Molecular Biology of HIV Replication) INSTRUCTOR'S GUIDE
Lane Recommended
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Please refer to the kit
insert for the Answers to
Study Questions
EDVO-Kit 335 Reverse Transcription PCR (RT-PCR): The Molecular Biology of HIV Replication) APPENDICES
Appendices
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APPENDICES Reverse Transcription PCR (RT-PCR): The Molecular Biology of HIV Replication) EDVO-Kit 335
Appendix A
EDVOTEK Troubleshooting Guides
After staining the gel, The gel was not stained for a sufficient
Repeat staining protocol.
the DNA bands are faint. period of time.
After staining, the ladder Wrong volumes of DNA and primer added Practice using micropipets
and control RT-PCR to RT-PCR reaction.
products are visible on the
gel but some student RT-PCR EdvoBead was shriveled or
samples are not present. RT-PCR EdvoBead had expired.
melted.
Low molecular weight Pipetting error-low amount of RNA Practice using micropipets.
band in RT-PCR samples template in RT-PCR sample.
To ensure adequate separation, make sure Be sure to run the gel the appropriate distance before staining
DNA bands were not
the tracking dye migrates at least 3.5 cm on and visualizing the DNA.
resolved.
7 x 7 cm gels and 6 cm on 7 x 14 cm gels.
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EDVO-Kit 335 Reverse Transcription PCR (RT-PCR): The Molecular Biology of HIV Replication) APPENDICES
Appendix B
Preparation and Handling of PCR Samples with Wax
ONLY For Thermal Cyclers WITHOUT Heated Lids, or Manual PCR Using Three Waterbaths
Using a wax overlay on reaction components prevents evaporation during the PCR process.
2. Centrifuge at full speed for ve seconds to collect sample at bottom of the tube.
3. Using clean forceps, add one wax bead to the PCR tube.
1. After PCR is completed, melt the wax overlay by heating the sample at 94 C for three minutes or until the wax
melts.
4. Use a pipette tip to puncture the thin layer of remaining wax. Using a fresh pipette tip, remove the PCR product
and transfer to a new tube.
5. Add 5 l of 10x Gel Loading Buffer to the sample. Proceed to Module II to perform electrophoresis.
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APPENDICES Reverse Transcription PCR (RT-PCR): The Molecular Biology of HIV Replication) EDVO-Kit 335
Appendix C
Bulk Preparation of Agarose Gels
To save time, the electrophoresis buffer and agarose gel solution can be prepared in larger quantities for sharing by
the class. Unused diluted buffer can be used at a later time and solidied agarose gel solution can be remelted.
Table
BULK ELECTROPHORESIS BUFFER Bulk Preparation of Electrophoresis Buffer
D
Quantity (bulk) preparation for 3 liters of 1x electro- Distilled Total Volume
50x Conc.
phoresis buffer is outlined in Table D. Buffer + Water Required
60 ml 2,940 ml 3000 ml (3 L)
BATCH AGAROSE GELS (1.0%)
Table
Bulk preparation of 1.0% agarose gel is outlined in Table E. Batch Preparation of
E 1.0% UltraSpec-Agarose
1. Use a 500 ml ask to prepare the diluted gel buffer
Amt of 50x Conc. Distilled Diluted
2. Pour the appropriate amount of UltraSpec-Agarose into the Agarose + Buffer + Water = Buffer (1x)
6. Dispense the required volume of cooled agarose solution for casting each Note:
gel. The volume required is dependent upon the size of the gel bed. QuickGuide instructions
and guidelines for casting
various agarose gels can be
7. Allow the gel to completely solidify. It will become rm and cool to the found our website.
touch after approximately 20 minutes. Proceed with electrophoresis (Mod- www.edvotek.com/
ule II) or store the gels at 4 C under buffer. quick-guides
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