sed
Revi nd
a
ated
Upd
Edvo-Kit #369
369
Human PCR
Toolbox
Experiment Objective:
In this experiment, students will isolate their DNA and use the Polymerase Chain
Reaction (PCR) with unique primers to amplify three different regions of their
genome. Results are analyzed using agarose gel electrophoresis.
See page 3 for storage instructions.
NOTE:
PCR Cycling Conditions have
changed. Please review
your PCR program before
performing the experiment.
369.150717
Human PCR Toolbox
EDVO-Kit
EDVO-Kit 369
369
Table of Contents
Page
3
Experiment Components
Experiment Requirements
4
Background Information
5
Experiment Procedures
Experiment Overview
Module I-A: Isolation of DNA from Human Cheek Cells
Module I-B: Isolation of DNA from Human Hair
Module II: Amplification of DNA Sequences
Module III: Separation of PCR Products by Electrophoresis
Module IV: Staining Agarose Gels
Study Questions
13
14
15
16
17
19
21
Instructor's Guidelines
Pre-Lab Preparations
Experiment Results and Analysis
Study Questions and Answers
22
23
27
30
Appendices
A EDVOTEK® Troubleshooting Guide
B Preparation and Handling of PCR Samples With Wax
C Bulk Preparation of Agarose Gels
33
34
36
37
Safety Data Sheets can be found on our website: www.edvotek.com
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Human PCR Toolbox
EDVO-Kit 369
369
EDVO-Kit
Experiment Components
Components
A PCR EdvoBeads™
Storage
Room Temp.
Check (√)
❑
Each PCR EdvoBead™ contains:
•
dNTP Mixture
•
Taq DNA Polymerase Buffer
•
Taq DNA Polymerase
•
MgCl2
•
Reaction Buffer
B-1
B-2
B-3
C
D
E
F
D1S80 Primer Mix concentrate
PV92 Primer Mix concentrate
Mitochondrial Primer Mix concentrate
EdvoQuick™ DNA ladder
Control DNA concentrate
TE Buffer
Proteinase K
-20° C Freezer
-20° C Freezer
-20° C Freezer
-20° C Freezer
-20° C Freezer
-20° C Freezer
Room Temp., desiccated
❑
❑
❑
❑
❑
❑
❑
This experiment is
designed for 25 human
DNA typing reactions.
All experiment components
are intended for educational
research only. They are not to
be used for diagnostic or drug
purposes, nor administered to
or consumed by humans or
animals.
NOTE: Components B-1, B-2, B-3, and D are now supplied in
concentrated form and require dilution prior to setting
up PCR reactions.
REAGENTS & SUPPLIES
Store all components below at room temperature.
Component
•
UltraSpec-Agarose™
•
Electrophoresis Buffer (50x)
•
10x Gel Loading Solution
• InstaStain® Ethidium Bromide
• FlashBlue™ Stain
• Snap-top Microcentrifuge Tubes
• Screw-top Microcentrifuge Tubes (1.5 ml - use for boiling)
• 0.2 ml PCR tubes
• Disposable plastic cups
• Salt packets
• 15 ml Conical tube
• Wax beads (for thermal cyclers without heated lid)
Check (√)
❑
❑
❑
❑
❑
❑
❑
❑
❑
❑
❑
❑
EDVOTEK, The Biotechnology Education Company, and InstaStain are registered trademarks of EDVOTEK, Inc.
EdvoBead, UltraSpec-Agarose, FlashBlue and EdvoQuick are trademarks of EDVOTEK, Inc.
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Human PCR Toolbox
EDVO-Kit
EDVO-Kit 369
369
Requirements
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
Thermal cycler (EDVOTEK Cat. # 532 highly recommended)
Horizontal gel electrophoresis apparatus
D.C. power supply
Balance
Microcentrifuge
Two Waterbaths for 55° C and 99°C Incubations (EDVOTEK Cat. #539 highly recommended)
UV Transilluminator or UV Photodocumentation system (use if staining with InstaStain® Ethidium Bromide)
UV safety goggles
White light visualization system (optional - use if staining with FlashBlue™)
Automatic micropipets (5-50 μl) with tips
Microwave, hot plate or burner
Pipet pump
250 ml flasks or beakers
Hot gloves
Disposable vinyl or latex laboratory gloves
Ice buckets and ice
Distilled or deionized water
Drinking Water (if isolating DNA from cheek cells)
Bleach solution
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Human PCR Toolbox
EDVO-Kit 369
369
EDVO-Kit
Background Information
VNTR HUMAN DNA TYPING
Polymorphic DNA refers to chromosomal regions that vary
widely from individual to individual. By examining several
of these regions within the genomic DNA obtained from
an individual, one may determine a "DNA Fingerprint" for
that individual. DNA polymorphisms are now widely used
for determining paternity/maternity, kinship, identification of human remains, and the genetic basis of various
diseases. The most far-reaching application, however, has
been in the field of criminal forensics. DNA from crime
victims and offenders can be matched to crime scenes,
often affecting the outcome of criminal and civil trials.
CRIME SCENE
Skin
Cells
SUSPECT #1
SUSPECT #2
Blood Draw
Blood Draw
The beginning of DNA fingerprinting occurred in the United
Kingdom in 1984, following the pioneering work of Dr.
Alex Jeffreys at the University of Leicester. Analysis by
Jeffreys led to the apprehension of a murderer in the first
DNA fingerprinting case in September 1987. The first U.S.
conviction occurred on November 6, 1987 in Orlando, FL.
Since then, DNA analysis has been used in thousands of
convictions. Additionally, over 100 convicted prison inmates have been exonerated from their crimes, including
several death row inmates.
In 1990, the Federal Bureau of Investigation (FBI) established the Combined DNA Index System (CODIS), a system
which allows comparison of crime scene DNA to DNA profiles in a convicted offender and a forensic (crime scene)
index. A match of crime scene DNA to a profile in the
convicted offender index indicates a suspect for the crime,
whereas a match of crime scene DNA to the forensic
index (a different crime scene) indicates a serial offender.
CODIS has now been used to solve dozens of cases where
authorities had not been able to identify a suspect for the
crime under investigation.
Body Fluid
Stain
Hair
Treat to release DNA
Perform PCR
to amplify
specific
polymorphic
regions
Crime Suspect Suspect
Scene
#2
#1
Suspect #2 matches
Crime Scene
Figure 1:
Extraction and Electrophoresis of DNA Samples.
The first step in forensic DNA fingerprinting is the collection of human tissue from the
crime scene or victim. These tissues include blood, hair, skin, and body fluids. The sample, often present as a stain, is treated with a detergent to rupture (lyse) cell membranes
and obtain DNA for further analysis (Figure 1). One early method, called Restriction
Fragment Length Polymorphism (RFLP) analysis, involves digesting DNA with restriction
enzymes, separating the fragments by agarose gel electrophoresis, transferring the DNA
to a membrane, and hybridizing the membrane with probes to polymorphic regions. This
method is statistically very accurate, but requires relatively large amounts of DNA and
takes several days to complete. Because of the time and DNA requirements, the RFLP
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EDVO-Kit 369
Human PCR Toolbox
method is no longer used in forensics, but remains in use
in certain medical genetics-based tests.
In forensics, the polymerase chain reaction (PCR) is now
used to amplify and examine highly polymorphic DNA
regions. These are regions that vary in length from
individual to individual and fall into two categories: 1)
Variable Number of Tandem Repeats (VNTR) and 2) Short
Tandem Repeats (STR). A VNTR is a region that is variably composed of a 15-70 base pair sequence, typically
repeated 5-100 times. An STR is similar to a VNTR except
that the repeated unit is only 2-4 nucleotides in length. By
examining several different VNTRs or STRs from the same
individual, investigators obtain a unique DNA fingerprint
for that individual which is unlike that of any other person
(except for an identical twin).
One VNTR known as D1S80, is present on chromosome 1
and contains a 16-nucleotide sequence which is variably
repeated between 16 and 40 times. An individual who
is homozygous for the D1S80 genotype will have equal
repeat numbers on both homologues of chromosome 1,
displaying a One VNTR known as D1S80, is present on
chromosome 1 and contains a 16-nucleotide sequence
which is variably repeated between 16 and 40 times. An
individual who is homozygous for the D1S80 genotype
will have equal repeat numbers on both homologues of
chromosome 1, displaying a single PCR product following
gel analysis (Figure 2A). More commonly, a person will be
heterozygous, with differing D1S80 repeat numbers. Amplification of DNA from heterozygous individuals will result
in two distinct PCR products (Figure 2B). For most applications, law enforcement agencies now use STRs as they are
more easily amplified and thus require less starting DNA.
The objectives of this experiment are to isolate human
DNA and compare DNA polymorphisms between individuals by PCR amplification and gel electrophoresis. In this
experiment, each student will 1) extract his/her DNA from
hair or cheek cells, 2) amplify DNA at the D1S80 locus by
PCR, and 3) examine the PCR products on agarose gels.
ALU-HUMAN TYPING
Figure 2A:
Homozygous
Condition
(Lane B) Maternal
Primer 1
Paternal
Primer 2
Primer 1
Paternal
Maternal
Figure 2B:
Heterozygous
Condition
(Lane C)
EdvoQuick™
DNA ladder
(Lane A)
Primer 2
A
B
C
2600bp
1400bp
1100bp
700bp
600bp
400bp
200bp
Gel results are not drawn to scale.
Figure 2:
PCR Amplification Products of D1S80
The human genome consists of 2.9 billion base pairs of
DNA. Of this total, only about 5% consists of exons which
code for protein. Introns and other noncoding sequences
make up the remainder; although some of these sequences may possess undiscovered
functions, most appear to have none. Many of these non coding sequences appear to be
self-replicating and are repeated hundreds or thousands of times throughout the genome.
These repetitive sequences have been termed “selfish” or “parasitic” DNA, as they often
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EDVO-Kit 369
Human PCR Toolbox
appear to possess no function except for their own reproduction. These repetitive elements account for more than
20 percent of the human genome.
Primer 1
Paternal
Maternal
Alu
u
Al
In 1979, it was discovered that human DNA contains a 300
base pair repetitive element. Copies of this element contain a recognition site for the restriction enzyme Alu I, and
were subsequently named Alu elements. Although Alu
elements have been found in exons, most exist in introns
and other non-coding regions. When Alu sequences do
insert into protein-coding regions, disruption of the gene
usually results, often causing harm to the organism. Alu
sequences replicate through an RNA intermediate which
is copied into a double-stranded DNA segment called a
retrotransposon. The details of this process are not well
understood. The retrotransposon then inserts elsewhere in
the genome. It is also theorized that most Alu sequences
are incapable of replication and that only a small number
of "master genes" are duplicated to form new elements.
Primer 2
Primer 1
Paternal
Maternal
One may test whether a person possesses an Alu insertion
at the PV92 locus by amplification of the locus using the
polymerase chain reaction (PCR). If a person is homozygous for the insertion, a gel of the PCR product will result
in a single band at 700 base pairs (Figure 3A). If a person
is heterozygous, i.e., possesses the insertion on one chromosome 16 homologue but not the other, two bands will
be present following PCR. One band will be 400 base pairs
and the other will be 700 base pairs (Figure 3B). If a person lacks the insertion on either chromosome homologue,
that person is said to possess the null genotype and PCR
will result in only one band at 400 base pairs (Figure 3C).
Alu
Although all humans (and other primates) possess hundreds of thousands of Alu elements, variations in their
placement may occur. DNA sequences which vary between
individuals are known as polymorphisms. For example,
one person may possess an Alu insertion at a specific DNA
locus, while another individual lacks that insertion. Furthermore, the insertion may be present or absent on each
homologous chromosome and are called dimorphic. One
such dimorphic Alu sequence is found on a section of chromosome 16 known as the PV92 locus. This section of DNA
is 700 nucleotides in length. Insertion of the 300 base pair
Alu element results in a length increase to 1000 base pairs.
Primer 2
Primer 1
Paternal
Maternal
Primer 2
Figure 3:
PV92 Locus on Chromosome 16.
To purify DNA for this type of analysis, almost any tissue or body fluid (except urine)
may be used. The most common sources of human DNA are hair and buccal cells (cheek
cells). Cells to be tested must be treated (lysed) to release their DNA into solution.
Following lysis, the cells are resuspended in a chelating agent, which removes cellular
cations that inhibit PCR.
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EDVO-Kit 369
Human PCR Toolbox
Porins
In this experiment, each student will extract his/her DNA
from hair or cheek cells and amplify DNA at the PV92 locus
by PCR. As a control, DNA purified from a cultured human
cell line may be used. The PCR product(s) will then be examined on agarose gels to determine whether the student
is homozygous (+/+), heterozygous (+/-), or null (-/-) for
an Alu insertion at the locus. Objectives of this experiment
are the isolation of human DNA and the comparison of DNA
polymorphisms between individuals by PCR amplification
and gel electrophoresis.
Cristae
Matrix
Ribosome
Inner membrane
Mitochondrial DNA
Figure 4:
Structure of a Mitochondrion.
MITOCHONDRIAL DNA TYPING
Mitochondria (plural for mitochondrion) are the
energy-producing organelles of the cell. Mitochondria are generally oblong or egg-shaped.
Both plant and animal cells possess mitochondria.
The number of mitochondria per cell varies depending on the cell type, ranging from only a few
in skin cells to thousands in skeletal muscle cells.
Unlike other organelles, mitochondria have two
separate membranes. The outer membrane is
fairly porous, possessing a protein called porin.
The inner membrane, however, is highly impermeable to ions and is enriched in a rare, negatively charged phospholipid known as cardiolipin.
The inner membrane is highly convoluted, with
infoldings called cristae (Figure 4) that greatly
increase the total membrane surface area. The
inner membrane also contains the enzymes that
catalyze cellular respiration, the process whereby
energy is produced for the cell.
Outer membrane
Fat & Sugar
Byproducts
Citric
Acid Cycle
Cytoplasm
Fatty
Acid
Oxidation
ATP
Synthase
Electron Transport Chain
Figure 5:
ATP
Energy for
Cell
Site of metabolic pathways that convert sugars and fats to energy.
The space inside the inner membrane is known as the matrix (Figure 4). Within the matrix
and inner membrane, the chemical reactions that produce energy for the cell take place.
As shown in Figure 5, sugars and fatty acids, broken down to two carbon units, enter a
series of reactions known as the citric acid or Krebs cycle. Sugars are broken down in the
cytoplasm while fatty acids are broken down in the mitochondria by a process known as ß
(beta) oxidation. The citric acid cycle generates electrons that enter the electron transport
chain, a cluster of protein complexes that reside in the inner membrane of the mitochondria. In the final step of energy production, protons generated by the electron transport
chain flow through a pump known as ATP synthase, driving the production of ATP, the primary energy-containing molecule used in biological systems. This final energy-producing
process is known as oxidative phosphorylation.
The DNA present in the matrix is distinct from the DNA found in the cell’s nucleus. Mitochondrial DNA (mtDNA) is contained in a single circular chromosome, shown in Figure 6,
that has been completely sequenced. The mitochondrial chromosome contains 16,569
base pairs of DNA and 37 genes. MtDNA encodes 13 polypeptides, all of which are sub-
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EDVO-Kit 369
Human PCR Toolbox
units of the respiratory chain comCy
toc
plex. This is shown on the map in
hro
me
D-Loop
Figure 6, illustrating the locations
l
B
a s
m
o ne
of genes that encode proteins in
s
0/16569
bo ge
complexes I and IV of the electron
Ri A
RN
transport chain. As shown, mtDNA
also encodes mitochondrial ribosomal RNA and the ATP synthase,
in addition to cytochrome B,
another constituent of the electron
12360
transport chain. MtDNA encodes
only part of the electron transport chain; nuclear DNA encodes
PCR
Complex I
the remaining complex subunits.
Products
11688
Genes
One peculiarity is that mitochondrial protein synthesis uses a
slightly different genetic code
9199
than cytoplasmic translation. As
8278
all cells possess only one nucleus
Co
m
but several hundred or thousand
Ge plex
ne IV
mitochondria, mtDNA is press
ent in great excess over nuclear
ATP
DNA in most cells. This relative
se
Syntha
abundance of mtDNA is taken
advantage of by forensic investiFigure 6: Genetic map of mitochondrial DNA.
gators after obtaining crime scene
specimens that are degraded or
otherwise insufficient for nuclear
DNA PCR analysis. The D-loop (Figure 6) has a
high degree of variability between individuals
and can be sequenced to demonstrate variations.
I
MtDNA typing, however, cannot be used to conclusively link suspects to crime scenes; rather, it
II
can be used to include or exclude suspects from
further scrutiny.
Complex I
Genes
III
During the past twenty years, an ever-increasing
number of diseases have been shown to be due
Figure 7: Patterns of inherited maternal mitochondrial diseases.
to mitochondrial dysfunction. These disorders result when mitochondrial ATP generation is insufficient to meet energy needs in a particular tissue. Because muscle and nerve cells contain large numbers of mitochondria, these organ
systems are most affected by mitochondrial dysfunction. Mitochondrial diseases may be
due to mutations in mtDNA genes or mutations in nuclear genes that encode mitochondrial enzymes. Diseases caused by mtDNA mutations include the myopathies, diseases
that affect various muscles and encephalomyopathies, which cause both muscular and
neurological problems. Huntington’s chorea, a devastating disease that results in dementia and loss of motor control, is caused by defects in oxidative phosphorylation and has
been mapped to a mutation in nuclear DNA encoding a non-mitochondrial protein. Other
diseases such as Alzheimer’s and Parkinson’s disease involve mitochondrial abnormalities,
although it is unclear how these abnormalities relate to disease pathology. Mitochondria
also appear to play roles in aging and in programmed cell death, also known as apoptosis.
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EDVO-Kit 369
Human PCR Toolbox
Since mitochondria are present in the cytoplasm, they are inherited independently from
the nucleus. A female egg cell possesses over
10,000 mitochondria, while a sperm cell has
very few. Thus during fertilization, mitochondrial DNA is inherited almost exclusively from
the mother. Although a small amount of paternal mtDNA is present in the fertilized egg, this
DNA appears to be selectively destroyed by the
newly fertilized egg. This pattern of inheritance
of mtDNA is known as maternal inheritance.
Maternal inheritance is indicated when all offspring, male and female, of the mother are afflicted with a specific condition (Figure 7). The
severity of any particular mitochondrial disorder
is highly variable, depending on the number
of mutated mitochondria inherited from the
mother (Figure 8).
POLYMERASE CHAIN REACTION
To examine polymorphisms, the Polymerase
Chain Reaction (PCR) is usually employed. PCR
was invented in 1984 by Dr. Kary Mullis at the
Cetus Corporation in California. The enormous
utility of the PCR method is based on its ease of
use and its ability to allow the amplification of
small DNA fragments. For this groundbreaking
technology, Mullis was awarded the Nobel Prize
in Chemistry in 1993.
nucleus
mutant
Mother with mild
or no symptoms
normal
number of
mitochondria
increases
Mature
oocyte
80%
mutant
50%
mutant
20%
mutant
+
+
+
Child with
severe
disease
Child with
mild
disease
Child with
no
disease
Fertilization
of oocyte
Figure 8: Mitochondrial inheritance
Before performing PCR, template DNA is extracted from various biological sources. Because
PCR is very sensitive, only a few copies of the
gene are required. Nevertheless, freshly isolated DNA will provide better amplification results
than older DNA specimens that may have become degraded. In order to amplify the specific
DNA or target sequence, two primers (short & synthetic DNA molecules) are designed to correspond to the ends of the target sequence.
To perform PCR, the template DNA and a molar excess of primers are mixed with the four
“free” deoxynucleotides (dATP, dCTP, dGTP, and dTTP), and a thermostable DNA polymerase.
The most commonly used DNA polymerase is Taq DNA polymerase. This enzyme, originally
purified from a bacterium that inhabits hot springs, is stable at very high temperatures. These
components (template DNA, primers, the four deoxynucleotides, and Taq DNA polymerase)
are mixed with a buffer that contains Mg+2, an essential cofactor for Taq polymerase. The PCR
reaction mixture is subjected to sequential heating/cooling cycles at three different temperatures in a thermal cycler.
•
In the first step, known as “denaturation”, the mixture is heated to near boiling (94° C 96° C) to “unzip” (or melt) the target DNA. The high temperature disrupts the hydrogen
bonds between the two complementary DNA strands and causes their separation.
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EDVO-Kit 369
Human PCR Toolbox
Target Sequence
= Separation of
two DNA strands
5'
3'
3'
5'
5'
3'
3'
5'
= Primer 1
Cycle 1
= Primer 2
5'
5'
3'
3'
3'
3'
5'
Anneal
2 primers
40°C - 65°C
5'
5'
3'
5'
5'
5'
3'
Cycle 3
Cycle 2
3'
5'
Denature
94°C
5'
3'
5'
5'
5'
5'
5'
5'
5'
3'
3'
5'
5'
3'
5'
5'
3'
5'
5'
5'
3'
Extension
72°C
3'
5'
5'
3'
5'
5'
3'
3'
5'
5'
3'
5'
5'
Figure 9:
Polymerase Chain Reaction
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Human PCR Toolbox
EDVO-Kit 369
•
In the second step, known as “annealing”, the reaction mixture is cooled to 40 - 65° C, which allows the primers to base pair with the target DNA sequence.
•
In the third step, known as “extension”, the temperature is raised to 72°C. This is the optimal temperature at
which Taq polymerase can add nucleotides to the hybridized primers to synthesize the new complementary
strands.
These three steps - denaturation, annealing, and extension - constitute one PCR “cycle”. Each PCR cycle doubles
the amount of the target DNA in less than five minutes. In order to produce enough DNA for analysis, twenty to
forty cycles may be required. To simplify this process, a specialized machine, called a “thermal cycler” or a “PCR
machine”, was created to rapidly heat and cool the samples.
In this experiment, students will extract their own DNA from hair or cheek cells. Using PCR, they will amplify DNA
at (1) two separate regions of the mitochondrial chromosomes, (2) the PV92 locus, and (3) the D1S80 region of
chromosome 1. As a control, DNA purified from a cultured human cell line may be used. The PCR product(s) will
then be analyzed using agarose gel electrophoresis.
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Human PCR Toolbox
EDVO-Kit 369
369
EDVO-Kit
Experiment Overview
EXPERIMENT OBJECTIVE:
In this experiment, students will isolate their DNA and use the Polymerase Chain Reaction
(PCR) with unique primers to amplify three different regions of their genome. Results are
analyzed using agarose gel electrophoresis.
LABORATORY SAFETY:
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.
• Contaminated laboratory waste (saliva solution, cup, pipet, etc.) must be disinfected with 15% bleach solution prior to disposal. Be sure to properly dispose
any biological samples according to your institutional guidelines.
Wear gloves
and safety goggles
Module I - 50 min.
Isolation of DNA from
Cheek Cells or Human Hair
Module II - 70-120 min.
Amplification of DNA
Sequences
Module III - 50-70 min.
LABORATORY NOTEBOOKS:
Separation of PCR Product
by Electrophoresis
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.
Module IV - 5-20 min.
Staining Agarose
Gels
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.
NOTE: Experimental times are
approximate.
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.
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Human PCR Toolbox
EDVO-Kit
EDVO-Kit 369
369
Module I-A: Isolation of DNA from Human Cheek Cells
Preferred Method
1.5 ml
2.
1.
4. SPIN
3.
T.C.
T.C.
60
T.C.
Full speed
2 min.
sec.
5.
Swirl
7.
6.
55° C
Vortex
or Flick
8.
99° C
15
15
140 µl
Lysis
Buffer
min.
min.
99
99
Warning!
Students should
use screw-cap
tubes when
boiling samples.
Vigorously 20 sec.
© 2013 Edvotek® All Rights Reserved.
9.
SPIN
10.
80 µl
Supernatant
Low speed
2 min.
1.
2.
LABEL a 1.5 ml screw top microcentrifuge tube and a cup with your lab group and/or initials.
RINSE your mouth vigorously for 60 seconds using 10 ml saline solution. EXPEL the
solution into cup.
STEP 4:
3. SWIRL the cup gently to resuspend the cells. TRANSFER 1.5 ml of solution into the
If cell pellet size is not
labeled tube.
large enough, repeat
steps 3 - 4 until you
4. CENTRIFUGE the cell suspension for 2 min. at full speed to pellet the cells. POUR off
have a large size pelthe supernatant, but DO NOT DISTURB THE CELL PELLET! Repeat steps 3 and 4 twice
let. For best results,
more.
make sure your cell
5. RESUSPEND the cheek cells in 140 μl lysis buffer by pipetting up and down or by vorpellet is at least the
size of a match head.
texing vigorously.
6. CAP the tube and PLACE in a waterbath float. INCUBATE the sample in a 55° C waterbath for 15 min.
7. MIX the sample by vortexing or flicking the tube vigorously for 20 seconds.
STEP 7:
If a vortex is not avail8. INCUBATE the sample in a 99° C waterbath for 15 min. Be sure to use screw-cap tubes
able, mix samples
when boiling DNA isolation samples.
by flicking the tube
9. CENTRIFUGE the cellular lysate for 2 minutes at low speed (6000 rpm).
vigorously for 20
10. TRANSFER 80 μl of the supernatant to a clean, labeled microcentrifuge tube. PLACE
seconds.
tube in ice.
11. PROCEED to Module II: Amplification of DNA Sequences.
OPTIONAL STOPPING POINT:
The extracted DNA may be stored at -20°C for amplification at a later time.
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Human PCR Toolbox
EDVO-Kit 369
369
EDVO-Kit
Module I-B: Isolation of DNA from Human Hair
1.
3.
2.
T.C.
4.
SPIN
5.
Tweeze
Full speed
10 sec.
Trim
6.
55° C
Vortex
or Flick
7.
8. SPIN
9.
55° C
15
15
min.
99
10.
99° C
Vigorously 20 sec.
Full speed
10 sec.
Vortex
or Flick
12. SPIN
11.
10
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
min.
99
Vigorously 20 sec.
IMPORTANT:
For best results, harvest
hairs from the scalp.
The root structure from
these hairs will be
thicker and will yield
more DNA than those
from the eyebrow.
Shaft
13.
80 µl
Supernatant
min.
99
Warning!
Students should
use screw-cap
tubes when boiling samples.
140 µl
Lysis
Buffer
Low speed
2 min.
HUMAN
HAIR
Sheath
Root
LABEL a 1.5 ml screw top microcentrifuge tube with your lab group and/or initials.
Using tweezers, GRASP 2-3 hair shafts at the base and PULL quickly. COLLECT at least 5 hairs that
include the root and the sheath (a sticky barrel-shaped layer of cells that encircles the root end of the
hair).
Using a clean scalpel or scissors, TRIM away any extra hair from the root (leave about 1 cm in length
from the root). TRANSFER the roots to the labeled tube using forceps.
CAP the tube and CENTRIFUGE the sample for 10 seconds at full speed to collect the roots at the bottom
of the tube.
ADD 140 μL lysis buffer to the tube. For best results, completely IMMERSE the follicles in the solution.
CAP the tube and PLACE it in a waterbath float. INCUBATE the sample in a 55° C waterbath for 15 min.
MIX the sample by vortexing or flicking the tube vigorously for 20 seconds.
STEPS 7 & 11:
CENTRIFUGE the sample for 10 seconds at full speed to collect the roots at the bottom
If a vortex is not
of the tube.
available, mix
INCUBATE the sample at 55° C for an additional 15 min.
samples by flicking
MOVE the sample to a 99° C waterbath. INCUBATE for 10 min. Be sure to use screwthe tube vigorously
for 20 seconds.
cap tubes when boiling samples.
MIX the sample by vortexing or flicking the tube vigorously for 20 seconds.
CENTRIFUGE the cellular lysate for 2 min. at low speed (6000 rpm).
TRANSFER 80 μl of the supernatant to a clean, labeled microcentrifuge tube. PLACE tube in ice.
PROCEED to Module II: Amplification of DNA Sequences.
OPTIONAL STOPPING POINT:
The supernatant may be stored at -20°C for amplification at a later time.
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Human PCR Toolbox
EDVO-Kit
EDVO-Kit 369
369
Module II: Amplification of DNA Sequences
1.
TC
3.
2.
• 20 µl Primer Mix*
• 5 µl Extracted DNA
• PCR EdvoBead
4. SPIN
Gently mix
TC
NOTES AND REMINDERS:
This kit includes enough
DNA for 5 control reactions.
At least one control reaction should be performed
per class to confirm that
PCR was successful.
TC
5.
6.
If your thermal cycler does
not have a heated lid, it is
necessary to overlay the
PCR reaction with wax to
prevent evaporation. See
Appendix B for guidelines.
TC
*Add one of the following primer solutions:
D1S80, PV92, or Mitochondrial
1.
LABEL a 0.2 ml PCR tube with the sample and your initials.
2.
ADD 20 μl Primer Mix (D1S80, PV92, or Mitochondrial), 5 μl extracted DNA (or control DNA) and the
PCR EdvoBead™ to the labeled 0.2 ml tube. At least one control reaction should be performed per
class to confirm that PCR was successful.
3.
MIX the PCR sample. Make sure the PCR EdvoBead™ is completely dissolved.
4.
CENTRIFUGE the sample for a few seconds to collect the sample at the bottom of the tube.
5.
AMPLIFY DNA using the following PCR cycling conditions:
D1S80
Initial Denaturation
Mitochondrial
94° C for 4 minutes
Denaturation
94° C for 30 sec.
94° C for 30 sec.
94° C for 60 sec.
Annealing
65° C for 30 sec.
65° C for 30 sec.
55° C for 60 sec.
Extension
72° C for 30 sec.
72° C for 60 sec.
72° C for 120 sec.
Number of Cycles
35
32
25
Final Extension
6.
PV92
72° C for 4 minutes
NOTE:
PCR Cycling Conditions
have changed. Please
review your PCR program before performing the experiment.
After PCR, ADD 5 μl of 10x Gel Loading Solution to the sample. PLACE tubes on ice.
PROCEED to Module III: Separation of PCR Products by Electrophoresis.
OPTIONAL STOPPING POINT:
The PCR samples may be stored at -20° C for electrophoresis at a later time.
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Human PCR Toolbox
EDVO-Kit 369
369
EDVO-Kit
Module III: Separation of PCR Products by Electrophoresis
1.
50
2.
3.
x
Concentrated
buffer
Distilled
water
1:00
Agarose
Flask
Caution! Flask will be HOT!
4.
60°C
WAIT
Pour
60°C
1.
2.
3.
4.
5.
6.
7.
7 x 14 cm gels are
recommended. Each gel
can be shared by 4-5 students. Place well-former
template (comb) in the
first set of notches.
If you are unfamiliar with
agarose gel prep and
electrophoresis, detailed
instructions and helpful
resources are available at
www.edvotek.com
5.
6.
IMPORTANT:
7.
20
min.
Wear gloves
and safety goggles
Table
DILUTE concentrated (50X) buffer with distilled
A Individual 1.5% UltraSpec-Agarose™ Gel
water to create 1X buffer (see Table A).
MIX agarose powder with 1X buffer in a 250 ml
Size of Gel
Concentrated
Distilled
TOTAL
Amt of
Casting tray Buffer (50x) + Water + Agarose =
Volume
flask (see Table A).
DISSOLVE agarose powder by boiling the solution.
7 x 7 cm
0.5 ml
24.5 ml
0.38 g
25 ml
MICROWAVE the solution on high for 1 minute.
7 x 14 cm
1.0 ml
49.0 ml
0.75 g
50 ml
Carefully REMOVE the flask from the microwave
and MIX by swirling the flask. Continue to HEAT
the solution in 15-second bursts until the agarose
is completely dissolved (the solution should be clear like water).
COOL agarose to 60° C with careful swirling to promote even dissipation of heat.
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.
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 solidifies.
REMOVE end caps and comb. Take particular care when removing the comb to prevent damage to the wells.
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EDVO-Kit 369
Human PCR Toolbox
Module III: Separation of PCR Products by Electrophoresis
8.
9.
Pour
1X Diluted
Buffer
11.
10.
Wear gloves
and safety goggles
Reminder:
Before loading the
samples, make sure
the gel is properly
oriented in the apparatus chamber.
8.
PLACE gel (on the tray) into electrophoresis chamber. COVER the gel with
1X electrophoresis buffer (See Table B for recommended volumes). The gel
should be completely submerged.
9. LOAD the entire volume (30 μl) into the well in
Table 1: Sample Table
the order indicated by Table 1.
10. PLACE safety cover. CHECK that the gel is propLane
Recommended
erly oriented. Remember, the DNA samples will
1
EdvoQuick™ DNA Ladder
migrate toward the positive (red) electrode.
2
Control DNA*
11. CONNECT leads to the power source and PERFORM
3
Student #1
electrophoresis (See Table C for time and voltage
4
Student #2
guidelines).
12. After electrophoresis is complete, REMOVE the gel
5
Student #3
and casting tray from the electrophoresis chamber
6
Student #4
and proceed to STAINING the agarose gel.
Sample Name
* Optional, or additional student sample.
Table
B
Table
1x Electrophoresis Buffer (Chamber Buffer)
C
Total Volume
Required
M6+
300 ml
6 ml
294 ml
125
M12
400 ml
8 ml
392 ml
70
M36
1000 ml
20 ml
980 ml
+
Distilled
Water
(1.5% - 7 x 14 cm Agarose Gel)
Recommended Time
Dilution
EDVOTEK
Model #
50x Conc.
Buffer
Time and Voltage Guidelines
Volts
150
Minimum
Maximum
45 min.
60 min.
55 min.
1 hour 15 min.
2 hours 15 min.
3 hours
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Human PCR Toolbox
EDVO-Kit 369
369
EDVO-Kit
Module IV-A: Staining Agarose Gels
with InstaStain® Ethidium Bromide
Preferred Method
1.
2.
3.
Moisten
the gel
4.
m
idiu
Eth
in®
Sta
sta
In
5.
4.
5.
6.
e
in
end
nt P
ate
.P
U.S
g
300 nm
3-5
min.
m Bromide
InstaStain® Ethidiu
U.S. Patent
2.
3.
STAIN
Ethid
nt Pending
U.S. Pate
1.
id
6.
-
InstaStain®
m
Bro
Pending
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. DO NOT STAIN GELS IN THE ELECTROPHORESIS APPARATUS.
MOISTEN the gel with a few drops of electrophoresis buffer.
Wearing gloves, REMOVE and DISCARD the clear plastic protective sheet from
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.
With a gloved hand, REMOVE air bubbles between the card and the gel by firmly
running your fingers over the entire surface. Otherwise, those regions will not
stain.
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® Ethidium Bromide card is in direct contact with the gel surface. STAIN
the gel for 3-5 minutes.
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.
Wear gloves and
UV safety goggles
BE SURE TO WEAR UV-PROTECTIVE EYEWEAR!
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Human PCR Toolbox
EDVO-Kit
EDVO-Kit 369
369
Module IV-B: Staining Agarose Gels
with FlashBlue™
1.
2.
10
x
Concentrated
FlashBlue™ Stain
Distilled
water
Wear gloves
and safety goggles
Flask
3.
5.
4.
STAIN
Pour
5
min.
(-)
1
2
3
4
5
6
DESTAIN
Pour
20
min.
(+)
1.
2.
3.
4.
5.
DILUTE 10 ml of 10x concentrated FlashBlue™ with 90 ml of water in a flask and MIX well.
REMOVE the agarose gel and casting tray from the electrophoresis chamber. SLIDE the gel off of the casting tray into a small, clean gel-staining tray.
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.
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 accelerate destaining.
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.
Alternate Protocol:
1.
2.
3.
DILUTE one ml of concentrated FlashBlue™ stain with 149 ml dH2O.
COVER the gel with diluted FlashBlue™ stain.
SOAK the gel in the staining liquid for at least three hours. For best results, stain gels overnight.
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EDVO-Kit 369
369
EDVO-Kit
Human PCR Toolbox
Study Questions
Answer the following study questions in your laboratory notebook or on a separate worksheet.
VNTR HUMAN DNA TYPING
1.
Compare your D1S80 PCR product with those of the rest of the class. Did any students have genotypes similar
to yours? How could you explain such similarities?
2.
What is polymorphic DNA? How is it used for identification purposes?
3.
What is CODIS? How is it used to solve crimes?
4.
What is an STR? A VNTR? Which (STR or VNTR) is predominantly used in law enforcement? Why?
ALU-HUMAN DNA TYPING
1.
Compare your Alu genotype with those of your classmates. Did anyone else have a similar result? If so, what
are some possible explanations?
2.
What is “selfish DNA”? How are Alu elements thought to replicate? What is the function(s) of Alu elements?
3.
Could dimorphic Alu elements be used for DNA identification (i.e., in criminal investigations)? Why or why not?
MITOCHONDRIAL DNA ANALYSIS
1.
What are the three energy-producing sets of chemical reactions that take place inside the mitochondrion?
2.
How are mitochondria different from other organelles inside the cell?
3.
Is it possible for a child to be healthy if his/her father is affected with a mitochondrial disease? From an unaffected mother? Why or why not? What might be some symptoms of such a disease?
4.
If a crime scene sample is too degraded for normal DNA profiling, are any further analyses possible? If so,
what assay(s) could be performed?
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Human PCR Toolbox
INSTRUCTOR'S GUIDE
EDVO-Kit 369
Instructor's Guide
NOTE:
OVERVIEW OF INSTRUCTOR’S PRELAB PREPARATION:
This section outlines the recommended prelab preparations and approximate time
requirement to complete each prelab activity.
Preparation For:
Module I:
Isolation of DNA
from Hair or
Cheek Cells
Module II:
Amplification of
DNA Sequences
What to do:
When:
PCR Cycling Conditions
have changed. Please
review your PCR program before performing the experiment.
Time Required:
Prepare and aliquot
various reagents
(Saline, Lysis buffer)
Up to one day before performing the
experiment. IMPORTANT: Prepare the Lysis
buffer no more than 30 minutes before
performing the experiment.
30 min.
Equilibrate waterbaths
at 55 ° C and boiling.
At least 30 min. before performing
the experiment.
15 min.
Prepare and aliquot various
reagents (Primer, DNA
template, ladder, etc.)
One day to 30 min. before performing
the experiment.
30 min.
Program Thermal Cycler
Anytime before performing
the experiment.
15 min.
Up to one hour before performing
the experiment.
45 min.
Module III:
Separation of PCR
Products by
Electrophoresis
Prepare diluted TAE buffer
Module IV:
Staining Agarose Gels
Prepare staining
components
Prepare molten agarose
and pour gel
Up to 10 min. before the class period.
10 min.
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EDVO-Kit 369
Human PCR Toolbox
INSTRUCTOR'S GUIDE
Pre-Lab Preparations
MODULE I-A : ISOLATION OF DNA FROM HUMAN CHEEK CELLS
Prepare the Saline Solution:
1.
To prepare the saline solution, dissolve all 8 salt packets in 500 ml of drinking
water. Cap and invert bottle to mix.
2.
Aliquot 10 ml of saline solution per cup. Distribute one cup per student.
Prepare the Lysis Buffer:
FOR MODULE I-A
Each Group should receive:
• One cup containing 10 ml
of saline solution
• One screw-cap tube
• One microcentrifuge tube
Reagents to be Shared by
Two Students:
• 300 μl Lysis buffer
• 15% bleach solution
(Prepared no more than 30 min. before starting the experiment.)
1.
Add 100 μl of TE buffer (E) to the tube of Proteinase K (F) and allow the sample
to hydrate for several minutes. After the sample is hydrated, pipet up and down
several times to thoroughly mix the material.
2.
Transfer the entire amount of the rehydrated Proteinase K solution to a 15 ml
conical tube containing an additional 4 ml of TE buffer (E).
3.
Invert the tube several times to mix. Label this tube “Lysis Buffer”. At this point,
the Lysis Buffer can no longer be stored. It should be used as soon as possible.
4.
Aliquot 300 μl of Lysis Buffer into 13 labeled microcentrifuge tubes.
5.
Distribute one tube of “Lysis Buffer” to each student pair.
Warning !!
Remind students to only
use screw-cap tubes when
boiling their DNA samples.
The snap-top tubes can
potentially pop open and
cause injury.
MODULE I-B: ISOLATION OF DNA FROM HUMAN HAIR
Preparation of Lysis Buffer
(Prepared no more than 30 min. before starting the experiment)
1.
Add 100 μl of TE buffer (E) to the tube of Proteinase K (F) and allow the sample
to hydrate for several minutes. After the sample is hydrated, pipet up and down
several times to thoroughly mix the material.
2.
Transfer the entire amount of the rehydrated Proteinase K solution to a 15 ml
conical tube containing an additional 4 ml of TE buffer (E).
3.
Invert the tube several times to mix. Label this tube “Lysis Buffer”. At this point,
the Lysis Buffer can no longer be stored. It should be used as soon as possible.
4.
Aliquot 300 μl of Lysis Buffer into 13 labeled microcentrifuge tubes.
5.
Distribute one tube of “Lysis Buffer” to each student pair.
FOR MODULE I-B
Each Group should receive:
• One screw-cap tube
• One microcentrifuge tube
Reagents to be Shared by
Two Students:
• 300 μl Lysis buffer
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Human PCR Toolbox
INSTRUCTOR'S GUIDE
EDVO-Kit 369
Pre-Lab Preparations
MODULE II: AMPLIFICATION OF DNA SEQUENCES
Preparation of the Primer
1.
Thaw the Primer Mix Concentrates (B-1, B-2, or B-3) on ice. B-1 corresponds to
the D1S80 Primers, B-2 corresponds to the PV92 Primers, and B-3 corresponds
to the Mitochondrial Primers.
2.
Add 1 ml of TE Buffer (E) to the tube of Primer Mix Concentrate. Cap tube and
mix.
3.
Aliquot 50 μl of the diluted Primer Mix into 13 labeled microcentrifuge tubes.
4.
Distribute one tube of diluted Primer Mix to each student pair.
FOR MODULE II
Each Student should receive:
• One PCR tube and PCR
EdvoBead™
• 20 μl Gel Loading Solution
Reagents to be Shared by
Two Students:
• 50 μl Primer Mix (D1S80,
PV92, or Mitochondrial)
Preparation of the Control DNA
1.
This kit includes enough DNA to set up 4 control reactions. At least one control reaction should be performed per class to confirm that PCR was successful.
2.
Thaw the tube of Control DNA Concentrate (D) on ice.
3.
Add 20 μl of TE buffer (E) to the tube containing Control DNA Concentrate. Pipet up and down to mix. This
is 1:3 diluted control DNA sample. Dispense 8 μl of this diluted control DNA for the D1S80 control reaction.
4.
To prepare control DNA for the other two primers (PV92, & Mitochondrial), take 10 μl of the 1:3 diluted
control DNA and dilute in 20 μl of TE buffer (E) – this is 1:9 diluted Control DNA.
5.
Dispense 8 μl of the 1:9 diluted control DNA for each PV92 and Mitochondrial control reaction.
6.
We recommend at least one control reaction per gel. Keep on ice.
Additional Materials:
•
Dispense 20 μl of 10x Gel Loading Solution to each student pair.
PCR Amplification
The Thermal cycler should be programmed as outlined in Module II in the Student’s
Experimental Procedure.
•
•
Accurate temperatures and cycle times are critical. A pre-run for one cycle (takes
approximately 3 to 5 min.) is recommended to check that the thermal cycler is
properly programmed.
For thermal cyclers that do not have a heated lid, it is necessary to place a layer
of wax above the PCR reactions in the microcentrifuge tubes to prevent evaporation. See Appendix B for instructions.
NOTE:
PCR Cycling Conditions
have changed. Please
review your PCR program before performing the experiment.
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EDVO-Kit 369
Human PCR Toolbox
INSTRUCTOR'S GUIDE
Pre-Lab Preparations
MODULE III: SEPARATION OF PCR PRODUCTS BY ELECTROPHORESIS
Preparation of Agarose Gels
This experiment requires one 1.5% agarose gel per 4-5 students. A 7 x 14 cm gel is
recommended. You can choose whether to prepare the gels in advance or have the
students prepare their own. Allow approximately 30-40 minutes for this procedure.
Individual Gel Preparation:
Each student group can be responsible for casting their own individual gel prior to
conducting the experiment. See Module III in the Student’s Experimental Procedure.
Students will need 50x concentrated buffer, distilled water and agarose powder.
Batch Gel Preparation:
To save time, a larger quantity of agarose solution can be prepared for sharing by
the class. See Appendix C.
Preparing Gels in Advance:
Gels may be prepared ahead and stored for later use. Solidified gels can be stored
under buffer in the refrigerator for up to 2 weeks.
Do not freeze gels at -20º C as freezing will destroy the gels.
Gels that have been removed from their trays for storage should be “anchored” back
to the tray with a few drops of molten agarose before being placed into the tray.
This will prevent the gels from sliding around in the trays and the chambers.
Additional Materials:
Each 1.5% gel should be loaded with the EdvoQuick™ DNA ladder and PCR reactions
from 4 or 5 students.
•
Aliquot 30 μl of the EdvoQuick™ DNA ladder (C) into labeled microcentrifuge
tubes and distribute one tube of EdvoQuick™ DNA ladder per gel.
NOTE:
Accurate pipetting is critical
for maximizing successful
experiment results. This
experiment is designed for
students who have had
previous experience with
micropipetting techniques
and agarose gel electrophoresis.
If students are unfamiliar
with using micropipets, we
recommended performing
Cat. #S-44, Micropipetting
Basics or Cat. #S-43, DNA
DuraGel™ prior to conducting this advanced level
experiment.
FOR MODULE III
Each Group should receive:
• 50x concentrated buffer
• Distilled Water
• UltraSpec-Agarose™
Powder
• EdvoQuick™ DNA ladder
(30 μl)
• Control PCR reaction
(optional)
NOTE:
QuickGuide instructions
and guidelines for casting
various agarose gels can be
found our website.
www.edvotek.com/
quick-guides
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Human PCR Toolbox
INSTRUCTOR'S GUIDE
EDVO-Kit 369
Pre-Lab Preparations
MODULE IV: STAINING AGAROSE GELS
InstaStain® Ethidium Bromide (PREFERRED METHOD)
InstaStain® Ethidium Bromide provides the sensitivity of ethidium bromide while
minimizing the volume of liquid waste generated by staining and destaining a gel.
An agarose 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.
FOR MODULE IV-A
Each Group should receive:
• 2 InstaStain® cards per
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 absent. 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 product. UV protective eyewear is required for visualization with a UV
transilluminator.
InstaStain® Ethidium Bromide cards and stained gels should be discarded using
institutional guidelines for solid chemical waste.
Wear gloves
and safety goggles
FlashBlue™
FlashBlue™ can be used as an alternative to Ethidium Bromide in this experiment.
However, FlashBlue™ is less sensitive than InstaStain® Ethidium Bromide and will
take a longer time to obtain results.
FlashBlue™ stain, however, is optimized to shorten the time required for both staining and destaining steps. Agarose gels can be stained with diluted FlashBlue™ for 5
minutes and destained for only 20 minutes. For the best results, leave the gel in liquid
overnight. This will allow the stained gel to “equilibrate” in the destaining solution,
resulting in dark blue DNA bands contrasting against a uniformly light blue background. A white light box (Cat. #552) is recommended for visualizing gels stained
with FlashBlue™.
•
•
FOR MODULE IV-B
Each Group should receive:
• 10 ml 10X concentrated
FlashBlue OR 100 ml
1x diluted FlashBlue
• Small plastic tray or weight
boat
• Distilled or deionized water
Stained gels may be stored in destaining liquid for several weeks with refrigeration, although the bands may fade with time. If this happens, re-stain the gel.
Destained gels can be discarded in solid waste disposal. Destaining solutions can
be disposed of down the drain.
Photodocumentation of DNA (Optional)
Once gels are stained, you may wish to photograph your results. There are many
different photodocumentation systems available, including digital systems that are
interfaced directly with computers. Specific instructions will vary depending upon the
type of photodocumentation system you are using.
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EDVO-Kit 369
Human PCR Toolbox
INSTRUCTOR'S GUIDE
Experiment Results and Analysis - VNTR Human DNA Typing
The results photo shows
an example of the possible
PCR products from different
genotypes. Students' PCR
products should show one
or two bands with lengths
between approx. 400 and
785 base pairs. The Control
DNA will have bands at approx. 420 bp and 600 bp.
Note – Depending on the PCR conditions used, a diffuse, small-molecular weight band, known as a "primer dimer", may be present below
the 200 bp marker. This is a PCR artifact and can be ignored. Other
minor bands may also appear due to nonspecific primer binding and
the subsequent amplification of these sequences.
D1S80 LOCUS
Forward
primer
(23 bp)
Variable region
Reverse
primer
(23 bp)
Constant
Constant
The PCR products generated by this experiment will range in size from 400 bp to 785 bp.
145 bp are constant between samples:
23 bp (Forward primer)
23 bp (Reverse primer)
99 bp (Flanks VNTR)
256 bp - 640bp are variable between samples, depending on VNTR number :16 bp per repeat
16 to 40 repeats present in the variable region
16 repeats times 16 bp per repeat equals 256 bp
40 repeats times 16 bp per repeat equals 640 bp
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Human PCR Toolbox
INSTRUCTOR'S GUIDE
EDVO-Kit 369
Experiment Results and Analysis - Alu Human DNA Typing
Note – Depending on the PCR conditions used, a diffuse, small-molecular weight band, known as a "primer dimer", may be present below
the 200 bp marker. This is a PCR artifact and can be ignored. Other
minor bands may also appear due to nonspecific primer binding and
the subsequent amplification of these sequences.
The results photo shows an example of the possible PCR products from different genotypes.
Lane
Recommended
Molecular Weight
---
Result
1
EdvoQuick™ DNA Ladder
2
Control DNA*
400 bp
Null for Alu insertion (-/-)
3
Student #1
700, 400 bp
Heterozygous for Alu insertion (+/-)
4
Student #2
700 bp
Homozygous for Alu insertion (+/+)
5
Student #3
700 bp
Homozygous for Alu insertion (+/+)
6
Student #4
700, 400 bp
Heterozygous for Alu insertion (+/-)
---
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EDVO-Kit 369
Human PCR Toolbox
INSTRUCTOR'S GUIDE
Experiment Results and Analysis - Mitochondrial DNA Analysis
Cy
toc
al
m s
so ne
bo ge
i
R A
RN
hro
me
D-Loop
B
0/16569
12360
PCR
Products
Complex I
Genes
Complex I
Genes
11688
9199
8278
Co
m
Ge plex
ne IV
s
Student's PCR products will show
two bands with lengths of 672
and 921 base pairs. The smaller
fragment corresponds to DNA from
two Complex I genes, whereas the
larger fragment corresponds to the
ATP synthase gene.
ATP
se
Syntha
Genetic Map of mitochondrial DNA
Note – Depending on the PCR conditions
used, a diffuse, small-molecular weight band,
known as a "primer dimer", may be present
below the 200 bp marker. This is a PCR artifact and can be ignored. Other minor bands
may also appear due to nonspecific primer
binding and the subsequent amplification of
these sequences.
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Human PCR Toolbox
INSTRUCTOR'S GUIDE
EDVO-Kit 369
Questions and Answers to Study Questions - VNTR Human DNA Typing
1. Compare your D1S80 PCR product with those of the rest of the class. Did any students have genotypes
similar to yours? How could you explain such similarities?
Each student’s PCR product should produce one or two bands of various sizes between 369 and 800 base pairs.
One band indicates that the student is homozygous for the D1S80 genotype, i.e. he/she possesses the same
number of copies of the 16 base pair repeat on each homologous chromosome. The presence of two bands
indicates that the student is heterozygous, with different repeat numbers on each chromosome. Identical
genotypes between students could indicate a common ancestry or similarities in race or ethnicity, although
any similarities are most likely coincidental.
2. What is polymorphic DNA? How is it used for identification purposes?
Polymorphic DNA refers to chromosomal regions that vary widely from person to person. This variation is usually in the length of a specific DNA region. By analyzing a number of these regions, one may obtain a “DNA
fingerprint” of a person that is extremely unlikely to match the DNA fingerprint of any other individual. DNA
fingerprinting is used for the identification of missing persons, human remains, and matching criminal suspects
to crime scenes.
3. What is CODIS? How is it used to solve crimes?
CODIS is an acronym for the Combined DNA Index System, a computer-based database containing DNA fingerprints. In the convicted offender database, DNA profiles of convicted felons are maintained. In the forensic
database, DNA fingerprints from crime scenes are maintained.
4. What is an STR? A VNTR? Which (STR or VNTR) is predominantly used in law enforcement? Why?
An STR is an acronym for a short tandem repeat, a DNA sequence of 2-4 base pairs that is repeated variably
from person to person. VNTRs, or variable number of tandem repeats, have longer repeat units of 15-70 base
pairs. STRs are now preferred to VNTRs as the length of their amplified products requires less template DNA,
often allowing even degraded samples to be amplified.
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Please refer to the kit
insert for the Answers to
Study Questions
INSTRUCTOR'S GUIDE
Human PCR Toolbox
EDVO-Kit 369
Questions and Answers to Study Questions - Mitochondrial DNA Analysis
1. What are the three energy-producing sets of chemical reactions that take place inside the mitochondrion?
The three energy-producing reaction cascades that occur in the mitochondrion are:
a) the citric acid, or Krebs, cycle
b) fatty acid (beta) oxidation; and
c) the electron transport chain (oxidative phosphorylation)
2. How are mitochondria different from other organelles inside the cell?
Mitochondria have a double membrane and also have their own unique DNA chromosome, RNA, and ribosomes.
3. Is it possible for a child to be healthy if his/her father is affected with a mitochondrial disease? From
an unaffected mother? Why or why not? What might be some symptoms of such a disease?
Mitochondrial diseases are inherited maternally, exclusively from the mother. Therefore, a child cannot inherit
a mitochondrial disease from an afflicted father. It is possible, however, for a child to be stricken with a mitochondrial disease inherited from a healthy mother who carries a small number of diseased mitochondria, if
these defective mitochondria are incorporated into the oocyte that becomes fertilized. Mitochondrial disorders
primarily affect the muscular and nervous systems, as these organs possess large numbers of mitochondria.
Typical symptoms of muscle disorders include muscle wasting and exercise intolerance. Nervous system disorders can result in headaches, seizures, and hearing and visual impairment.
4. If a crime scene sample is too degraded for normal DNA profiling, are any further analyses possible? If
so, what assay(s) could be performed?
If a crime scene specimen is too degraded for standard DNA fingerprinting (which is performed on nuclear
DNA) it may still be possible to analyze the mitochondrial DNA. This further analysis is possible because most
cells typically possess many copies of mitochondrial DNA but only one nuclear DNA copy.
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EDVO-Kit 369
Human PCR Toolbox
APPENDICES
Appendices
A
EDVOTEK® Troubleshooting Guide
B
Preparation and Handling of PCR Samples With Wax
C
Bulk Preparation of Agarose Gels
Material Safety Data Sheets:
Now available for your convenient download on www.edvotek.com
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Human PCR Toolbox
APPENDICES
EDVO-Kit 369
Appendix A
EDVOTEK® Troubleshooting Guides
DNA EXTRACTION
PROBLEM:
There is no cell pellet
after centrifuging the
cheek cell suspension.
I was not able to extract
DNA from hair.
Poor DNA Extraction
The extracted DNA is
very cloudy.
CAUSE:
ANSWER:
Not enough cheek cells in suspension
Mouth must be vigorously rinsed for at least 60 sec.
to harvest loose cheek cells.
Sample not centrifuged fast enough
Spin cells at maximum speed (17,000 x g) for 2 min.
If your centrifuge does not reach this speed, spin at
highest available speed for 4 min.
Not enough hairs used for extraction
Use at least five hairs for the DNA extraction.
No follicle was present on hair shaft
The best place to collect hairs for this experiment is
the head. Pick hair follicles which have a bulbous
base (sheath cells).
Samples not mixed well enough during
extraction
In addition to flicking the tube, vortex or pipet up and
down to mix the sample.
Proteinase K inactive because it was
prepared too far in advance.
Prepare Proteinase K within one hour of use.
Water baths not at proper temperature
Use a thermometer to confirm water bath set point.
Not enough DNA
Try cheek cell extraction. Final DNA concentrations
are usually higher.
Cellular debris from pellet transferred to
tube
Centrifuge sample again and move supernatant to a
fresh tube. Take care to avoid pellet.
Cellular debris not separated from
supernatant
Centrifuge sample again. If possible, centrifuge at a
higher speed. Move cleared supernatant to a fresh
tube.
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EDVO-Kit 369
Human PCR Toolbox
APPENDICES
Appendix A
EDVOTEK® Troubleshooting Guides
PCR AND ELECTROPHORESIS
PROBLEM:
CAUSE:
ANSWER:
Make sure the heated lid reaches the appropriate temperature.
Sample has evaporated
There is very little liquid
left in tube after PCR
If your thermal cycler does not have a heated lid, overlay the
PCR reaction with wax (see Appendix B for details)
Make sure students close the lid of the PCR tube properly.
Pipetting error
Make sure students pipet 20 µL primer mix and 5 µL extracted
DNA into the 0.2 mL tube.
Ensure that the electrophoresis buffer was correctly diluted.
The ladder, control DNA,
and student PCR
products are not visible
on the gel.
The gel was not prepared properly.
Gels of higher concentration (> 0.8%) require special attention
when melting the agarose. Make sure that the solution is
completely clear of “clumps” and glassy granules before
pouring gels.
The proper buffer was not used for gel preparation.
Make sure to use 1x Electrophoresis Buffer.
The gel was not stained properly.
Repeat staining.
Malfunctioning electrophoresis unit or
power source.
Contact the manufacturer of the electrophoresis unit
or power source.
After staining the gel,
the DNA bands are faint.
The gel was not stained for a sufficient
period of time.
Repeat staining protocol.
After staining the gel,
the gel background is
very dark.
The gel needs to be destained longer.
Submerge the gel in distilled or deionized water. Allow the
gel to soak for 5 minutes.
Student DNA sample was not
concentrated enough.
Poor DNA extraction. Repeat Module I (Isolation of
DNA from Human Cheek Cells)
Student DNA sample was degraded.
If DNA is not used right after extraction, store sample at -20°C.
Wrong volumes of DNA and primer added
to PCR reaction.
Practice using micropipets
Some student samples
have more/less
amplification than others.
Concentration of DNA varies by sample.
There is an inherent variability in the extraction process.
Low molecular weight
band in PCR samples
Primer dimer
Low concentration of extracted DNA in PCR reaction.
DNA bands were not
resolved.
To ensure adequate separation, make sure
Be sure to run the gel the appropriate distance before staining
the tracking dye migrates at least 3.5 cm on and visualizing the DNA.
7 x 7 cm gels and 6 cm on 7 x 14 cm gels.
DNA bands fade when
gels are kept at 4°C.
DNA stained with FlashBlue™ may
fade with time
After staining, the ladder
and control PCR products
are visible on the gel but
some student samples
are not present.
Re-stain the gel with FlashBlue™
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Human PCR Toolbox
APPENDICES
EDVO-Kit 369
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.
How to Prepare a Wax overlay
1.
Add PCR components to the 0.2 ml PCR Tube as outlined in Module II.
2.
Centrifuge at full speed for five seconds to collect sample at bottom of the tube.
3.
Using clean forceps, add one wax bead to the PCR tube.
4.
Place samples in PCR machine and proceed with Module II.
Preparing PCR Samples for Electrophoresis
1.
After PCR is completed, melt the wax overlay by heating the sample at 94° C for three minutes or until the wax
melts.
2.
Using a clean pipette, remove as much overlay wax as possible.
3.
Allow the remaining wax to solidify.
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 μl of 10x Gel Loading Buffer to the sample. Proceed to Module III to perform electrophoresis.
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EDVO-Kit 369
Human PCR Toolbox
APPENDICES
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 solidified agarose gel solution can be
remelted.
Table
Bulk Electrophoresis Buffer
D
Quantity (bulk) preparation for 3 liters of 1x electrophoresis buffer is outlined in Table D.
Bulk Preparation of Electrophoresis Buffer
50x Conc.
Buffer
60 ml
BATCH AGAROSE GELS (1.5%)
Bulk preparation of 1.5% agarose gel is outlined in Table E.
1.
Use a 500 ml flask to prepare the diluted gel buffer
Distilled
Water
Total Volume
Required
2,940 ml
3000 ml (3 L)
+
Table
Batch Preparation of
1.5% UltraSpec-Agarose™
E
Amt of
Agarose
2.
Pour the appropriate amount of UltraSpec-Agarose™ into the
prepared buffer. Swirl to disperse clumps.
3.
With a marking pen, indicate the level of solution volume on
the outside of the flask.
4.
Heat the agarose solution as outlined previously for individual gel preparation. The heating time will require adjustment due to the larger total
volume of gel buffer solution.
5.
Cool the agarose solution to 60°C with swirling to promote even dissipation
of heat. If evaporation has occurred, add distilled water to bring the solution up to the original volume as marked on the flask in step 3.
6.
Dispense the required volume of cooled agarose solution for casting each
gel. The volume required is dependent upon the size of the gel bed.
7.
Allow the gel to completely solidify. It will become firm and cool to the
touch after approximately 20 minutes. Proceed with electrophoresis (Module II) or store the gels at 4º C under buffer.
+
50x Conc.
Buffer
+
Distilled
Water
=
Diluted
Buffer (1x)
4.5 g
6.0 ml
294 ml
300 ml
6.0 g
8.0 ml
392 ml
400 ml
60˚C
Note:
The UltraSpec-Agarose™ kit
component is usually labeled
with the amount it contains.
Please read the label carefully. If the amount of agarose is not specified or if the
bottle's plastic seal has been
broken, weigh the agarose
to ensure you are using the
correct amount.
NOTE:
QuickGuide instructions
and guidelines for casting
various agarose gels can
be found our website.
www.edvotek.com/
quick-guides
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