Southern Blot

The Biotechnology Education Company
EDVOTEK, Inc. 1-800-EDVOTEK www.edvotek.com

EVT 2012_04_12AM
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DNA Fingerprinting III:
Southern Blot Analysis
Storage: See Page 3 for
specic storage instructions
EXPERIMENT OBJECTIVE:
This experiment introduces students to Southern Blot
analysis used in DNA Fingerprinting for a hypothetical
paternity determination.
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The Biotechnology Education Company 1-800-EDVOTEK www.edvotek.com
DNA Fingerprinting III: Southern Blot Analysis
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Experiment
Page
Experiment Components 3
Experiment Requirements 4
Background Information 5
Experiment Procedures
Experiment Overview and General Instructions 13
Module I: Agarose Gel Electrophoresis 14
Module II: Southern Blot Transfer 16
Module III: Non-Isotopic Detection of DNA 19
Study Questions 22

Instructor's Guidelines
Notes to the Instructor 24
Pre-Lab Preparations 27
Experiment Results and Analysis 29
Study Questions and Answers 30
Appendices
A Agarose Gel Preparation For Southern Blot Analysis 32
B Quantity Preparations for Agarose Gel Electrophoresis 33

C Staining and Visualization of DNA with
InstaStain Ethidium Bromide Cards 34
Material Safety Data Sheets 35
Table of Contents
All 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.
EDVOTEK and The Biotechnology Education
Company are registered trademarks of
EDVOTEK, Inc.
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Experiment
EDVOTEK - The Biotechnology Education Company
1.800.EDVOTEK www.edvotek.com
FAX: 202.370.1501 email: info@edvotek.com
Experiment Components

Storage
Biotin labeled samples, ready for electrophoresis Store components
A-I in the freezer.
A Standard DNA fragments
B Mother DNA cut with Enzyme 1
C Mother DNA cut with Enzyme 2
D Child DNA cut with Enzyme 1
E Child DNA cut with Enzyme 2
F Father 1 DNA cut with Enzyme 1
G Father 1 DNA cut with Enzyme 2
H Father 2 DNA cut with Enzyme 1
I Father 2 DNA cut with Enzyme 2
Reagents for Southern Blot and Non-Isotopic Detection
J Detection buffer, 20x concentrated Refrigerator
K Shielding Buffer Refrigerator
L Termination buffer, 20x concentrated Refrigerator
M NBT/BCIP Tablets Freezer
N Streptavidin-Alkaline Phosphatase, SAAP Freezer
Other Reagents & Supplies Room temp
UltraSpec-Agarose powder
Concentrated electrophoresis buffer
Nylon Membranes (7 x 14 cm)
Precut lter paper (7 x 14 cm)
STORAGE OF PERISHABLES
This experiment includes perishable components which were sent on wet ice.
Store these components at -20C (-4F). Please note what type of freezer you
have and store components accordingly.
Frost-free Freezer
Most refrigerator/freezers in homes are frost free. This means the freezer
goes through warming cycles to eliminate frost (defrost cycle). If using this
type of freezer, keep the enzymes in the foam chest (with the ice brick) in
which they were sent. This will help maintain the enzymes at -20C when
the freezer goes through the defrost cycle.
Non Frost-free Freezer
These older model freezers, which are still sold but are harder to nd, do
not go through warming cycles. Therefore, ice will build up on freezer walls
over time. If using this type of freezer, check to make sure that it maintains
temperature at -20C.
This experiment
contains enough
reagents for 5
groups.
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Requirements
Horizontal gel electrophoresis apparatus
D.C. power supply
Automatic micropipets with tips
Balance
UV Transilluminator (for optional staining)
Incubator (37 C, 65 C, and 80 C)
Microwave, hot plate or burner
100 ml graduated cylinder
10 ml graduated cylinder, or 5 or 10 ml pipets and pumps
250 ml asks or beakers
Hot gloves or beaker tongs
Safety goggles and disposable laboratory gloves
Plastic wrap
Paper towels
Forceps
Plastic trays (non-acrylic, to hold 7 x 14 cm membrane)
Distilled or deionized water (approximately 12-15 liters)
Ice (for pre-lab)
NaCl
NaOH
Concentrated HCl
5
Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/laboratory use only.
This document, or any part, may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK, Inc.
Copyright 1996-2012 EDVOTEK, Inc., all rights reserved. EVT 2012_04_12AM
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DNA Fingerprinting
DNA ngerprinting (also called DNA typing) allows for the unambiguous identication
of the source of DNA. The method has become very important in forensic laboratories
where it is used to provide evidence in paternity and criminal cases. In contrast to the
more conventional procedures, such as blood typing that can only exclude a suspect, DNA
ngerprinting can provide positive identication with great accuracy.
When rst introduced, DNA ngerprinting involved the electrophoretic analysis of DNA
fragment sizes generated by restriction enzymes. With the advent of the Polymerase
Chain Reaction (PCR), specic regions of DNA are amplied and analyzed by gel electro-
phoresis or other procedures.
This experiment will focus on restriction enzymes and Southern blot analysis as applied to
DNA ngerprinting.
ABOUT RESTRICTION ENZYMES
Restriction enzymes are endonucleases that catalyze the cleavage of phosphodiester
bonds within both strands of DNA. They require Mg
+2
for activity and generate a 5 prime
(5) phosphate and a 3 prime (3) hydroxyl group at the cleavage sites. The distinguish-
ing feature of restriction enzymes is that they cleave DNA at very specic base sequences
that are recognition sites. Restriction enzymes are produced by many different species of
bacteria (including blue-green algae). Over 3,000 restriction enzymes have been discov-
ered and catalogued.
Restriction enzymes are named according to the organism from which they are isolated.
This is done by using the rst letter of the genus followed by the rst two letters of the
species. Only certain strains of particular species produce restriction enzymes that follow
the species designation in the name. Roman numerals are used to designate the different
restriction enzymes produced by the same organism.
Specic double-stranded DNA sequences (usually 4 to 8 base pairs in length) are used by
restriction enzymes as recognition sites. Cleavage occurs within or near these sites that
are indicated by arrows (see examples below). Recognition sites are usually symmetrical,
i.e., both DNA strands in the site have the same base sequence when read 5 to 3.

5'-GAATTC-3' > 5'-G AATTC-3'
3'-CTTAAG-5' > 3-CTTAA G-5'

As shown, Eco RI causes staggered cleavage at its site. The resulting ends of DNA frag-
ments are called sticky or cohesive due to the single-stranded regions at the ends
that are complementary.
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DNA Fingerprinting Analysis
Certain restriction enzymes, such as Hae III, introduce cuts that are directly opposite each other.
This type of cleavage generates blunt ends.

5'-GGCC-3' > 5'-GG CC-3'
3'-CCGG-5' > 3'-CC GG-5'

The recognition sites of another subset of restriction enzymes contain variable base positions.
For example, Ava I recognizes:

5'-CPyCGPuG-3' (Py = pyrimidine = C or T and
3'-GPuGCPyC-5' Pu = purine = G or A)

Keep in mind that A pairs with T and G pairs with C. Consequently, there are four possible
sequences Ava I recognizes. Recognition sites of this type are called degenerate recognition
sites.
Another group of restriction enzymes have recognition sites that are divided by a certain num-
ber of totally variable bases. For example, Bgl I recognizes:

5'-GCCNNNNNGGC-3' (N = A, G, C or T)
3'-CGGNNNNNCCG-5'

There are 625 possible sequences that Bgl I can cleave. The only bases the enzyme truly recog-
nizes are the six G-C anking base pairs at both ends of the palindrome. The Bgl I recognition
site is hyphenated and is separated by 5 base pairs.
The size of the DNA fragments generated by restriction enzyme cleavage depends on the
distance between recognition sites. In general, bigger DNA affords a greater probability that
a given recognition site will occur. The number of times a restriction enzyme cleaves double-
stranded DNA is determined as 4
N
where N is the number of bases that make up the restriction
enzyme recognition site. For Eco RI, N = 6 bases, therefore 4
6
= 4096. This number designates
the frequency that Eco RI will cut DNA.
ABOUT AGAROSE GEL ELECTROPHORESIS
Agarose gel electrophoresis is a procedure used to analyze DNA fragments generated by
restriction enzymes. Agarose gels consist of microscopic pores that act as a molecular sieve.
Samples of DNA are loaded into wells formed in the gel during gel casting. Since DNA has a
strong negative charge at neutral pH, it migrates through the gel towards the positive elec-
trode. In general, DNA fragments are separated by the gel according to their size. The smaller
the fragment, the faster it migrates. After electrophoresis, DNA fragments can be visualized
by staining the gel with dyes. Restriction enzyme cleavage of relatively small DNA molecules,
such as plasmids and viral DNAs, usually produce discrete banding patterns of the DNA frag-
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ments. However, cleavage of large DNA, such as human chromosomal DNA, generates
many differently sized fragments that exceed the resolving capacity of agarose gels.
Consequently, the cleaved DNA is visualized as a smear after staining and has no obvious
banding patterns.
ABOUT SOUTHERN BLOT ANALYSIS
Southern blots facilitate RFLP analysis of complex DNA. After electrophoresis, the gel is
sequentially treated in HCl and NaOH. The HCl treatment introduces apurinic sites in DNA
that makes phophodiester bonds at these sites labile and introduces nicks in double-
stranded DNA. Apurinic sites result when a purine base (adenine) is cleaved as in an A/T
base pair. The NaOH treatment disrupts hydrogen bonds between the base pairs. The
sequential acid and base treatments generate small single stranded DNA fragments from
large DNAs . This facilitates transfer of DNA fragments from the gel to the nylon mem-
brane.
In Southern blots a replica of the gel DNA bands is transferred to a nylon membrane.
This is done by placing the nylon membrane on the gel after electrophoresis and transfer-
ring the fragments to the membrane by capillary action or by electrotransfer. Transferred
DNA becomes permanently adsorbed to the nylon and can be manipulated easier than a
gel.
Analysis of transferred DNA requires hybridization of the specic gene(s) with probe(s)
that are small single stranded piece of DNA. Initially radioactively labeled probes where
used for analysis, but more recently (as in this experiment ) non-isotopic detection systems
are routinely used. In forensic RFLP analysis the probes are single stranded DNA frag-
ments that contains base sequences that are complementary to the variable arrays of
tandem repeats found in human chromosomes. A solution containing the probe is incu-
bated with the membrane that contains the single stranded transferred DNA fragments.
Under the proper conditions, the probe will only base pair (hybridize) to DNA fragments
that contain the complementary sequences. The nylon membrane used in Southern blot
analysis is washed to remove excess probe. Only fragments that are hybridized to the
probe will be detected as discrete DNA bands that serve as a set of ngerprints.
ABOUT DNA FINGERPRINTING
No two individuals have exactly the same pattern of restriction enzyme recognition sites.
This is due to the large number of alleles present in human DNA . Alleles are alternate
forms of a gene that result in alternative expressions of genetic traits. Chromosomes occur
in matching pairs, one of maternal and the other of paternal origin. The two copies of a
gene at a given chromosomal locus represent a composite of parental genes that consti-
tute part of an individuals unique genotype. It follows that alleles have differences in
their base sequences that result in frequency variations of restriction enzyme recognition
sites. Other differences in base sequences between individuals can occur because of mu-
tations and deletions that create or eliminate restriction recognition sites. Figure 1 shows
how a silent mutation that can eliminate a restriction enzyme recognition site without
altering the protein product.
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No two individuals have exactly the same pattern of restriction enzyme recognition sites. This
is due to the large number of alleles present in human DNA . Alleles are alternate forms of a
gene that result in alternative expressions of genetic traits. Chromosomes occur in matching
pairs, one of maternal and the other of paternal origin. The two copies of a gene at a given
chromosomal locus represent a composite of parental genes that constitute part of an indi-
viduals unique genotype. It follows that alleles have differences in their base sequences that
result in frequency variations of restriction enzyme recognition sites. Other differences in
base sequences between individuals can occur because of mutations and deletions that create
or eliminate restriction recognition sites. Figure 1 shows how a silent mutation that can
eliminate a restriction enzyme recognition site without altering the protein product.
TGTTTA|TGTTTA|TGTTTA|.........variable number
Eco RI site
5' ACG AAT TCC 3' Coding DNA
2
H N Thr - Asn - Ser COOH Protein
5' ACG AAC TCC 3' Codon changed
by mutation
Thr - Asn - Ser COOH Protein
2
H N
Figure 1: Effect of a point mutation results in an altered amino acid code.
When tandum arrays are anked
by restriction recognition sites,
the length of the repeat will
determine the size of the restric-
tion enzyme fragment generated.
Variations in the length of these
fragments between individu-
als in a population are known
as restriction fragment length
polymorphisms (RFLP). RFLPs are
a manifestation of the unique
molecular genetic prole, or
ngerprint, of an individuals
DNA. As shown in Figure 2, there
are different sizes of repetitive
sequences.
In forensic, DNA samples are extracted and puried from small specimens of skin, blood, se-
men, or hair roots collected at the crime scene and are routinely amplied by the polymerase
chain reaction (PCR) . Small amounts of DNA suitable for analysis can also be obtained from
dried stains of semen or blood. Prior to the discovery of PCR, the restriction enzyme, Hae III
was used for DNA ngerprinting analysis. RFLP analyses was performed on DNA samples col-
lected at the crime scene and were compared to DNA ngerprints obtained from suspects. If
RFLP patterns matched it was reasonable to assume that the suspect was at the crime scene.
In practice, thirteen different probes containing various repetitious sequences are used in
order to satisfy certain statistical criteria for positive identication. In addition, the same DNA
sample is digested with a different restriction enzymes and probed to rmly establish an indi-
viduals unique DNA ngerprint..
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= repetitious sequence array (variable length)
larger
smaller
( ) ) (
= recognition site
Restriction
Fragments
Allele 2 Allele 1
Figure 2
Decreasing
Fragment
Size
1 2 3 4
Figure 3
Lane 1 Mother
Lane 2 Child
Lane 3 Father
Lane 4 Unrelated
For paternity determinations, DNA is obtained from the mother,
child, and possible fathers. A childs DNA is a composite of both
parent DNA. Therefore, comparison of DNA fragmentation pat-
terns obtained from the mother and child will give a partial match.
DNA bands in the childs ngerprint that are not present in the
mothers prole must match with those from the biological father.
Because of allelic differences, not all of the bands present in the
parents ngerprint will appear in the childs ngerprint. However,
as shown in Figure 3, DNA bands that appear in the childs nger-
print must be found in the ngerprint from either the father or
mother.
ABOUT THE POLYMERASE CHAIN REACTION
The Polymerase Chain Reaction (PCR) has two important advantages over
RFLP-based DNA ngerprinting analysis. The rst is the sensitivity of
PCR that allows for DNA ngerprinting identication using much smaller
amounts of DNA. The second advantage is the speed of PCR analysis, which
allows critical questions to be answered quickly as compared to Southern
Blot analysis.
PCR amplication requires the use of a thermostable DNA polymerase, such
as Taq polymerase. Puried from a bacterium known as Thermus Aquaticus
that inhabits hot springs, Taq polymerase is commonly used in PCR because
it remains stable at near-boiling temperatures. Also included in the PCR
reaction are the four deoxynucleotides (dATP, dCTP, dGTP, and dTTP) and
two synthetic oligonucleotides primers, typically 15-30 base pairs in length.
These components, together with the DNA to be amplied, are incubated in
an appropriate buffer that contains Mg
+2
. The primers are designed to cor-
respond to the DNA to be amplied also referred to as the target. The PCR
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reaction mixture is subjected to sequential heating/cooling cycles at three different tempera-
tures in a thermal cycler (Figure 4).
In the rst step, the template is heated to near boiling (94 C - 96 C.) to denature (melt)
the target DNA. This step, known as denaturation disrupts the hydrogen bonds be-
tween the two complimentary DNA strands and causes their separation.
In the second step, the reaction mixture is cooled to a temperature that is typically in
the range of 45 C- 65 C. In this step, known as annealing, the two primers that are in
great excess to the template, bind to the separated DNA strands.
In the third step, known as extension, the temperature is raised to 72C. At this tem-
perature the Taq polymerase is maximally active and adds nucleotides to the two hybrid-
ized primers to synthesize the new complimentary strands.
DNA ngerprinting analysis has become increasingly signicant in court cases involving mur-
der, rape, physical battery, and other types of crimes. Jurors are often asked to determine the
validity of DNA evidence, resulting in both acquittals and convictions of suspected criminals.
To ensure greater accuracy, scientists have incorporated standardization procedures in DNA n-
gerprint analysis. Standard DNA fragments are used to determine the exact size of individual
DNA fragments in a DNA ngerprint. It is generally accepted that DNA ngerprints are identi-
cal only in the case of identical twins.
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3' 5'
3' 5'
5' 3'
5' 3'
5'
5' 3'
3' 5'
5' 3'
5'
5'

Denature 94C
5'
Extension
72C
3' 5'
Separation of
2 DNA strands
=
Primer 1 =
Primer 2 =

5' 3'
5'
Anneal
2 primers
45C
3' 5'
5'
5'
5'
3' 5'
5'
5'
5' 3'
5'
5'
5'
5' 3'
5' 3'
5' 3'
5' 3'
5' 3'
5' 3'
5'
5' 3'
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1

C
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2

C
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3

Target Sequence
5' 3'
5' 3'
5' 3'
Figure 4: Polymerase Chain Reaction
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ABOUT THIS EXPERIMENT
The objective of the experiment is to match DNA ngerprinting patterns and identify the bio-
logical father. In an actual DNA based paternity determination, biotinylated probes would be
used to detect complementary DNA sequences transferred to the membrane. The detection
system employed would be identical to that used in this experiment.
In this experiment predigested and biotinylated DNA will be used to facilitate classroom in-
struction of Southern blots. In the hypothetical paternity case DNA from the mother, child and
two possible fathers were extracted from blood samples. DNA samples were cleaved under
the same conditions with two restriction enzymes in separate reactions.
.
The following are the experiment steps:
1. Biotin labeled precut DNA samples are separated by gel electrophoresis.
2. DNA fragments are transferred to a nylon membrane by Southern blot analysis. Biotin
groups will serve as labels for the transferred DNA.
3. To detect the presence of the biotinylated DNA a two step procedure will be employed. In
the rst step, non specic sites on the membrane will be blocked.
4. For the second step, a covalently linked streptavidin-alkaline phosphatase complex (SAAP)
will be incubated with the nylon membrane. The SAAP will bind to the biotinylated DNA.
Binding to DNA occurs by the streptavidin-biotin interaction. Since the alkaline phospha-
tase is covalently linked to the streptavidin, it will also bind to the biotinylated DNA.
5. The membrane is incubated with a substrate for alkaline phosphatase, 5-Bromo-4-chloro-
3-indolyl phosphate (BCIP). This reaction causes Nitro Blue tetrazolium (NBT) to become
reduced. Upon reduction NBT is converted into an insoluble blue product that precipi-
tates on the nylon membrane indicating the position of the transferred DNA.
There is no blood or human tissues used in this experiment.
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EXPERIMENT OBJECTIVE:
This experiment introduces students to Southern Blot analysis used in DNA Fingerprinting
for a hypothetical paternity determination.
LABORATORY SAFETY
1. Gloves and goggles should be worn routinely as good laboratory practice.
2. Exercise extreme caution when working with equipment that is used in con-
junction with the heating and/or melting of reagents.
3. DO NOT MOUTH PIPET REAGENTS - USE PIPET PUMPS.
4. Exercise caution when using any electrical equipment in the laboratory.
5. Always wash hands thoroughly with soap and water after handling reagents or bio-
logical materials in the laboratory.
LABORATORY NOTEBOOK RECORDINGS:
Address and record the following in your laboratory notebook or on a separate work-
sheet.
Before starting the Experiment:
Write a hypothesis that reects the experiment.
Predict experimental outcomes.
During the Experiment:
Record (draw) your observations, or photograph the results.
Following the Experiment:
Formulate an explanation from the results.
Determine what could be changed in the experiment if the experiment were
repeated.
Write a hypothesis that would reect this change.
Experiment Overview and General Instructions
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AGAROSE GEL REQUIREMENTS FOR THIS EXPERIMENT
Recommended gel size: 7 x 14 cm
Number of sample wells required: 10
Agarose gel concentration: 0.8%
PREPARING THE AGAROSE GEL
1. Close off the open ends of a clean and dry gel bed (casting tray) by using rubber
dams or tape.
2. Place a well-former template (comb) in the rst set of notches at the end of the
bed. Make sure the comb sits rmly and evenly across the bed.
3. To a 250 ml ask or beaker, add agarose powder and buffer as indicated in the
Reference Tables (Appendix A) provided by your instructor. Swirl the mixture to
disperse clumps of agarose powder.
4. With a marking pen, indicate the level of the solution volume on the outside of
the ask.
5. Heat the mixture using a microwave oven or burner to dissolve the agarose pow-
der.
6. Cool the agarose solution to 60C with careful swirling to promote even dissipa-
tion of heat. If detectable evaporation has occurred, add distilled water to bring
the solution up to the original volume marked in step 4.
After the agarose is cooled to 60C:
7. Place the bed on a level surface and pour the cooled agarose solution into the
bed.
8. Allow the agarose to completely solidify. It will become rm and cool to the
touch after approximately 20 minutes.
9. After the gel is solidied, be careful not to damage or tear the wells while re-
moving the rubber dams or tape and comb(s) from the gel bed.
10. Place the gel (on its bed) into the electrophoresis chamber, properly oriented,
centered and level on the platform.
11. Fill the electrophoresis apparatus chamber with the appropriate amount of
diluted (1x) electrophoresis buffer (refer to Table B on the instruction sheet from
the Appendix provided by your instructor).
Module I: Agarose Gel Electrophoresis
If you are unfamiliar with
agarose gel preparation and
electrophoresis, detailed
instructions and helpful
resources are available at
www.edvotek.com
Important Note
Continue heating until
the nal solution ap-
pears clear (like water)
without any undissolved
particles. Check the
solution carefully. If you
see particles, the agarose
is not completely dis-
solved.
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LOADING THE SAMPLES
Make sure the gel is completely submerged under buffer before loading
the samples and conducting electrophoresis.
Gel with 2 rows, 6 wells per row:
(Model #M12 with two standard 6-tooth combs)
Load 35-38 l of each sample.
First Row
Lane Tube
1 A Standard DNA fragments
2 B Mother DNA cut with Enzyme 1
3 C Mother DNA cut with Enzyme 2
4 D Child DNA cut with Enzyme 1
5 E Child DNA cut with Enzyme 2
Second Row
Lane Tube
2 F Father 1 DNA cut with Enzyme 1
3 G Father 1 DNA cut with Enzyme 2
4 H Father 2 DNA cut with Enzyme 1
5 I Father 2 DNA cut with Enzyme 2
RUNNING THE GEL
1. After the DNA samples are loaded, properly orient the cover and
carefully snap it onto the electrode terminals.
2. Insert the plugs of the black and red wires into the corresponding
inputs of the power source.
3. Set the power source at the required voltage and conduct electro-
phoresis for the length of time determined by your instructor.
4. Check to see that current is owing properly - you should see
bubbles forming on the two platinum electrodes.
5. After the electrophoresis is completed, disconnect the power and remove the gel
from the bed. (See Appendix C for optional staining step.)
6. Proceed to Module II - Soutnern Blot Analysis.
Module I: Agarose Gel Electrophoresis
Reminder:
Before loading the samples,
make sure the gel is
properly oriented in the
apparatus chamber.
+
Black Red
Sample wells
Electrophoresis can be completed

in 15-20 minutes under optimal
conditions. For Time and Voltage
recommendations, refer to Table C
(from Appendix A).
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Quick Reference:
The depurination procedure must
be brief (no longer than 8 minutes).
Prolonged exposure to HCl com-
pletely depurinates DNA strands.
Subsequent treatment with a dena-
turation solution would fragment
the completely depurinated DNA
molecules into very short oligo-
nucleotides, which are poor targets
for probes.
During this procedure, the bro-
mophenol blue tracking dye in the
gel will change color from blue to
greenish blue to yellow. After 8
minutes, the dye should be greenish
to slightly yellow in color.
OVERVIEW
During this procedure you will perform the Southern blot, which
involves the transfer of DNA fragments from the agarose gel to a
nylon membrane. After the transfer, the membrane will be baked
for a short time to x the DNA to the membrane.
After electrophoresis, the gel is treated in HCl and NaOH. The HCl
treatment introduces apurinic sites in DNA which makes phophodi-
ester bonds at these sites labile and therefore introduces nicks in
double-stranded DNA. Apurinic sites result when the purine base is
removed, such as an adenine residue from the A/ T base pair. The
NaOH treatment disrupts the the interstrand hydrogen bonds be-
tween the base pairs. The sequential acid and base treatments re-
sult in the formation of small fragments from large DNA fragments.
This facilitates the transfer of DNA fragments to the nylon mem-
brane. This procedure also causes double-stranded DNA restriction
fragments to be converted into their single-stranded form.
Module II: Southern Blot Analysis
DEPURINATION/DENATURATION
(Approximately 1 hour)
1. After electrophoresis, place one agarose gel per tray containing 200 ml of 0.25 N HCl at
room temperature. Allow the depurination to proceed for a maximum of 8 minutes. Stop
depurination if the dye becomes completely yellow before 8 minutes.
2. Carefully discard the HCl solution - do not reuse.
3. Rinse the gel with several changes of 500 ml distilled or deionized water.
4. Add 200 ml of DNA denaturation solution (0.5M NaOH/0.6M NaCl) and soak the agarose
gel for 15 minutes.
Because of the density of the solution, the gel will oat, so periodically shake the tray to
immerse the gel in solution.
5. Discard the denaturation solution and add a fresh second 200 ml of DNA denaturation
solution.
6. Continue to soak the gel for 30 minutes.
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SETTING UP THE SOUTHERN BLOT TRANSFER
(Approximately 30 minutes)
6. Place a sheet of plastic wrap on a at level lab bench.
7. Remove the gel from the tray when the second 30 minute incubation in the
NaOH/NaCl denaturation solution is complete. Save this solution to wet the
nylon membrane in step 11 below.
8. Place the gel (well side down) directly on the plastic wrap. By inverting the
gel, the smooth surface is on top for contact with the membrane.
9. Wearing gloves and using forceps, trim the nylon membrane to the size of the
gel. If your gel is 7 x 14 cm, no trimming is necessary.
Note: The membranes are very thin white sheets that may be sandwiched between one
or two sheets of protective covering. If you are not sure which is the membrane, check
with your instructor.
10. Carefully pick up a membrane at the edges with two forceps.
11. Slightly bend the membrane in the middle and slowly wet the membrane,
from the middle toward the edges, in the DNA Denaturation Solution which is
in the tray from step 7.
12. Release the membrane and gently submerge. Allow it to become thoroughly
saturated with DNA Denaturation Solution for 5 minutes.
13. Remove the saturated membrane from the DNA Denaturation Solution and
place it on top of the inverted agarose gel.
14. Place a piece of lter paper, which has been cut to the same size as
the membrane, on top of the membrane.
15. Roll a 5 or 10 ml pipet across the lter paper. This will remove air
bubbles.
16. Carefully place a stack of paper towels approximately 1.5 to 2 inches
thick on top of the lter paper.
17. Place an empty 400 ml beaker in an empty tray on top of the paper
towels for weight. Do not use anything heavier than an empty beaker to
provide weight.
18. Allow the blot to progress 3-4 hours or overnight.
WEAR GLOVES - DO
NOT TOUCH THE
NYLON MEMBRANE
WITH BARE HANDS.
Areas of the membrane
touched by ungloved
hands will leave oil resi-
dues and will not bind
DNA during transfer.
Many gloves contain
powder, which will
increase the background
on the membrane. Put
on gloves and wash
them under tap water
to remove any residual
powder. Handle the
membrane with clean
forceps.
Gel
Nylon
membrane
Filter paper
Paper towels
OPTIONAL STOPPING POINT
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19. After incubating 3-4 hours or overnight, remove the tray, beaker, and all the paper towels.
20. Wearing rinsed gloves and using forceps, ip the stack (paper towels and lter paper) over
to lie on the paper towels.
21. Using a pencil, draw through each sample well and trace its position on the nylon mem-
brane and identify lane one.
22. Using forceps, remove the membrane from the gel. Note the thickness and consistency of
the dehydrated gel.
The gel can be discarded since all further processing takes place with the nylon membrane.
23. Take the membrane and lay it on a dry paper towel with the DNA side up (the side of the
membrane which was in direct contact with the gel).
24. Using a pencil, label the DNA side of the membrane with your lab group number or initials
onto the corner of the membrane. The bromophenol blue dye can be seen on the mem-
brane.
25. For optimal results, place the membrane between two small sheets of lter paper and
place into an 80
o
C oven for 30 minutes.
26. Proceed to the non-isotopic detection procedure.
OPTIONAL STOPPING POINT

The dried membrane can be stored at room temperature, away from moisture and between two
sheets of lter paper until you are ready to continue.
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Module III: Non-isotopic Detection of DNA
NON-ISOTOPIC DETECTION OF DNA
(Approximately 4 hours)
During this experiment, you will process the membrane to visualize DNA bands. There
are no stopping points in this module.
MEMBRANE SHIELDING
The purpose of this procedure is to shield non-specic binding sites on the nylon mem-
brane. This helps to reduce the overall background. Use only one membrane per tray
during processing.
1. Rehydrate the baked membrane for 5 minutes in 100 ml of diluted Detection Buffer
(J), in a tray. Keep the DNA side of the membrane UP. Place only one membrane per
tray.
2. While the membrane is rehydrating, warm 50 ml of Membrane Shielding buffer (K) to
65C in the incubation oven or on a hot plate.
Use a graduated cylinder to measure 50 ml of Shielding Buffer (K) and place into
a 400 ml beaker.
Place in the 65C incubation oven, or exercise caution if warming the solution on
a hot plate set at 65
C.
Do not allow the temperature to exceed 65
C. If the temperature exceeds 65
C,
the solution may become thickened (like gelatin) and difcult to disperse evenly
over the membrane.
3. Stabilize the membrane with forceps. Pour and discard the Detection buffer from the
tray. Do not allow the membrane to drop into the sink.
4. Slowly add 50 ml of the warmed Membrane Shielding Buffer (K), to the tray, pour-
ing the buffer very gently over the top of the membrane. The membrane should still
have the DNA side facing up.
5. Cover the tray with the lid and place into a 60 C - 65
C incubation oven for 1 hour.

(NOTE: Temperature should not exceed 65 C.)
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DETECTION
6. After the one-hour incubation, remove the lid from the tray and place it upside down on
the lab bench.
7. Turn down the oven to 37
C for color development which begins with step 18.

8. Using forceps to hold the membrane in the tray, carefully pour off the Shielding buffer.
9. Place the membrane on the underside of the tray lid, set aside.
10. Rinse out the inside of the tray with distilled water. Shake out the tray to remove excess
water. Do not dry with paper towels (lint may increase background).
11. Place the membrane back into the tray, DNA side up. Use only one membrane per tray!
12. In a clean 50 ml Erlenmeyer ask, add 8 microliters of SAAP (N) to 10 ml of diluted Detec-
tion buffer. Mix well. Cover the membrane in the tray with this solution.
13. Incubate at room temperature for 10 minutes. Occasionally rock the tray from side to side
to ensure that the membrane is covered with solution.
14. Remove the membrane from the tray and place it on the underside of the lid, as before.
15. Wash out the tray thoroughly with distilled water. Remove any excess water.
16. Place the membrane back into the tray, DNA side up. Rinse off the lid with distilled water.
17. Wash the membrane extensively to remove any unbound
and non-specically bound SAAP. First, wash in 400 ml of
diluted Detection Buffer for 20 minutes with occasional
rocking. Discard the solution.
18. Second, wash in 200 ml of diluted Detection Buffer for 15
minutes with occasional rocking. Discard the solution.
19. Third, wash in 200 ml of diluted Detection Buffer for 15 minutes with occasional rocking.
Prepare color development solution during this wash. Discard the solution.
20. Ten minutes before the completion of the third membrane wash, prepare the Color Devel-
opment solution.
Dissolve the BCIP/NBT tablets in exactly 40 ml of distilled water.
Vortex until completely dissolved.
This is enough solution for all ve groups and must be used within one hour.
Step 17:
A shaking platform can be
used to rock the tray and
disperse the wash solution.
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COLOR DEVELOPMENT
21. After the incubation from step 19 is complete, carefully remove the mem-
brane from the tray and place it on the underside of the tray lid.
22. Add 8 ml of complete Color Development solution to the tray.
23. Place the membrane DNA SIDE DOWN into the tray and cover with a lid
or plastic wrap. Make sure the membrane is in complete contact with the
color development solution.
24. Place the tray inside a 37
o
C incubation oven in the dark. Allow the reac-
tion to proceed for several hours.
25. To determine the extent of the reaction, you should examine the mem-
brane after 30 to 60 minutes. Blue bands corresponding to the position of the DNA
fragments should be clearly visible. If all of the predicted bands are visible, proceed
to the termination phase (step 27). If the bands are not visible, continue the incuba-
tion.
26. Re-examine after 3 to 4 hours. All the bands should be visible. If you have trouble
visualizing any of the bands, a prolonged development time is permissible. However,
the background on the membrane may become darker. If any bands can be seen,
even though they may be faint, it is recommended that you proceed to Termination
(step 27).
TERMINATION
27. Place the membrane on the tray lid. Completely discard the Color Development solu-
tion and thoroughly rinse the tray with distilled water.
25. Transfer the membrane to the rinsed tray. Soak the membrane in 100 ml of diluted
Termination solution (L) for 5 minutes. Protect the membrane from strong direct
light.
26. Allow the membrane to dry in the dark between 2 sheets of lter paper at room tem-
perature. Alternatively, dry for 5 minutes in a 80
o
C oven.
27. After recording the results, the membrane can be stored in a dark place for several
months. Prolonged exposure to bright light will increase the background.
Steps 21-24 Alternative:
Use a sealable, leak-proof
bag to place the membrane
in for color development.
Add the complete color de-
velopment solution, remove
any air pockets, and seal the
bag. Place it on a tray in
the incubation oven.
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Answer the following study questions in your laboratory notebook or on a
separate worksheet.
1. Why do different individuals such as siblings have different restriction
enzyme recognition sites?
2. What is the function of probes in DNA paternity analysis?
3. Why is there more than one single locus probe used in an actual pater-
nity DNA test?
4. What advantage does Southern Blot Analysis offer for genetic testing?
Study Questions
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M
o
n
- Fri 9 a
m

-

6

p
m

E
T
(1-800-338-6835)
E
D
V
O
-
T
E
C
H SERVIC
E
1-800-EDVOTEK
Mon - Fri
9:00 am to 6:00 pm ET
FAX: 202.370.1501
Web: www.edvotek.com
email: info@edvotek.com
Please have the following
information ready:
Experiment number and title
Kit lot number on box or tube
Literature version number
(in lower right corner)
Approximate purchase date
Technical Service
Department
Instructors Guide
Class size, length of laboratory sessions, and availability of equipment are factors
which must be considered in the planning and the implementation of this experiment
with your students. These guidelines can be adapted to t your specic set of circum-
stances. If you do not nd the answers to your questions in this section, a variety of
resources are continuously being added to the EDVOTEK web site. In addition, Techni-
cal Service is available from 9:00 am to 6:00 pm, Eastern time zone. Call for help from
our knowledgeable technical staff at 1-800-EDVOTEK (1-800-338-6835).
EDUCATIONAL RESOURCES
Electrophoresis Hints, Help and Frequently Asked Questions
EDVOTEK Experiments are designed for maximum success in the classroom setting.
However, even the most experienced students and teachers occasionally encounter
experimental problems or difculties. The EDVOTEK web site provides several sugges-
tions and reminders for conducting electrophoresis, as well as answers to frequently
asked electrophoresis questions.
Online Ordering
now available
Visit our web site for information
about EDVOTEKs complete line
of hands-on experiments for
biotechnology and biology education.
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Notes to the Instructor:
APPROXIMATE TIME REQUIREMENTS
Module I
The experiment begins with agarose gel electrophoresis of biotin-labeled
samples, ready for electrophoresis. The total time for Module 1 is approxi-
mately 3 hours.
Module II
The Southern blot should be set up immediately following electrophoresis.
It will require two-days: 2 hours on Day 1 to prepare the blot which can
progress overnight. On Day 2, the blot is disassembled and the membrane
is baked for 30 minutes in an oven.
Module III
This is the non-isotopic detection phase of the experiment. Once detection
begins it must be completed during the same time period. It will require ap-
proximately 4 hours.
Pre-lab preparations
Pre-lab preparations and dispensing of biologicals and reagents take approx-
imately 1-2 hours.
Agarose Gel preparation
Whether you choose to prepare the gel(s) in advance or have the students
prepare their own, allow approximately 30-40 minutes for this procedure.
Generally, 20 minutes of this time is required for gel solidication. See sec-
tion Options for Preparing Agarose Gels below.
Quick Reference:
Approximate
Time Requirements
1. Agarose Gel
Electrophoresis
3 hours

2. Southern Blot
Transfer
Day 1: 2 hours
Day 2: 45 min.
3. Non-isotopic
Detection of DNA
4 hours
Time and Voltage
Recommendations
Minimum / Maximum
Volts
150
125
70
50
15 / 20 min
20 / 30 min
35 / 45 min
50 / 80 min
Table
C
EDVOTEK Electrophoresis Model
M6+ M12 & M36
Minimum / Maximum
25 / 35 min
35 / 45 min
60 / 90 min
95 / 130 min
Conducting Electrophoresis
The approximate time for electrophoresis will vary from
15 minutes to 2 hours. Generally, the higher the voltage
applied, the faster the samples migrate. However, depend-
ing upon the apparatus conguration and the distance
between the two electrodes, individual electrophoresis
units will separate DNA at different rates. Follow manufac-
turer's recommendations. Time and Voltage recommenda-
tions for EDVOTEK equipment are outlined in Table C.
OPTIONS FOR PREPARING AGAROSE GELS
A minimum of 10 sample wells per gel are required. If using the EDVOTEK
Model #M12 electrophoresis unit, cast a 7 x 14 cm gel with two 6-well combs.
Alternatively, use a 10-well comb accessory (Double Comb, Cat. # 683).
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There are several options for preparing agarose gels for the experiment.
1. Individual Gel Casting:
Each student lab group can be responsible for casting their own individual gel prior
to conducting the experiment.
2. Preparing Gels in Advance:
Gels may be prepared ahead and stored for later use. Solidied gels can be stored
under buffer in the refrigerator for up to 2 weeks.
Do not store gels at -20C. 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 hot, molten agarose before placing the gels into
the apparatus for electrophoresis. This will prevent the gels from sliding around in
the trays and the chambers.
3. Batch Gel Preparation:
A batch of agarose gel can be prepared for sharing by the class. To save time, a
larger quantity of UltraSpec-Agarose can be prepared for sharing by the class. See
instructions for "Batch Gel Preparation".
GEL CONCENTRATION AND VOLUME
The agarose gel concentration required for this experiment is 0.8% weight by volume.
OPTIONAL GEL STAINING AFTER ELECTROPHORESIS
Optional gel staining can be performed with InstaStain Ethidium
Bromide (Cat. #2001, not included). InstaStain EtBr.
1. Stain the gel according to InstaStain product instructions (Ap-
pendix C). If photographic documentation equipment is avail-
able, photograph the stained gel for a permanent record.
2. After viewing and/or photography of the stained gel, proceed to
the Southern blot.
Notes to the Instructor:
Caution: Ethidium Bromide is a listed
mutagen. Disposal of the InstaStain
EtBr cards, which contain only a few
micrograms of ethidium bromide, is
minimal compared to the large volume
of liquid waste generated by traditional
ethidium bromide staining procedures.
Disposal of InstaStain cards and gels
should follow institutional guidelines for
chemical waste.
Note: The DNA samples have been intentionally spiked with chromosomal DNA to show a
smeared pattern when stained with Ethidium bromide. Specic probed bands will be visible
on the membrane once the DNA has been transferred and the membrane has been pro-
cessed with the detection agents.
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Pre-Lab Preparations
MODULE I: AGAROSE GEL ELECTROPHORESIS
The experiment begins with agarose gel electrophoresis of biotin-labeled samples, ready for
electrophoresis.
A Standard DNA fragments
B Mother DNA cut with Enzyme 1
C Mother DNA cut with Enzyme 2
D Child DNA cut with Enzyme 1
E Child DNA cut with Enzyme 2
F Father 1 DNA cut with Enzyme 1
G Father 1 DNA cut with Enzyme 2
H Father 2 DNA cut with Enzyme 1
I Father 2 DNA cut with Enzyme 2
Note: The membranes are
very thin white sheets that
may be sandwiched between
one or two sheets of printed
or plain protective covering. If
you are not sure which sheet
is the membrane, please call
EDVOTEK Technical Service
(1-800-338-6835).
MODULE II: SOUTHERN BLOT ANALYSIS
General Preparations (for 5 blots)
1. For each gel, gather the following:
1 piece of pre-cut nylon membrane (cut to t your gel size)
1 piece of pre-cut blotting lter paper (cut to t your gel size)
20-30 paper towels (towels should be large enough to cover the gel)
2. On day two of the Southern Blot transfer, set up an 80C oven.
Preparation of Solutions

1. Prepare 1.0 liter of approximately 0.25 N HCl. Mix together:
21 ml Concentrated HCl (12 N)
979 ml Distilled/deionized water
2. Prepare 2.0 liters of the alkaline/salt DNA denaturation solution,
0.5 M NaOH/0.6 M NaCl.

1.8 L Distilled or deionized water
40.0 g NaOH pellets
70.0 g NaCl
Add NaOH and NaCl to the water. Use a magnetic stir plate to dissolve.
Add distilled water to a nal volume of 2.0 liters.
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Pre-Lab Preparations
MODULE III: NON-ISOTOPIC DETECTION OF DNA
Once detection begins it must be completed during the same time period. It will require
approximately 4 hours. DO NOT MIX THE SAAP SOLUTION (N) OR THE NBT/BCIP TABLETS
(M) UNTIL INDICATED IN THE EXPERIMENT. All other required buffers can be prepared
prior to the lab.
1. Prepare the Detection Buffer solution from the 20X concentrated stock ( J) by mixing
the following components:
250.0 ml 20x concentrated Detection Buffer (J)
4.75 L Distilled or deionized water.
Mix well. Each blot will require 0.9 L of the diluted Detection Buffer.
2. The Shielding Buffer (K) is not concentrated, and should be used without dilution.
Do not warm it until just before you need it. Prepare just enough for the blot which
is ready for processing. Detailed instructions for warming are outlined in the Stu-
dent Experiment Procedure.
3. Prepare the Termination Solution from 20x concentrated Termination Solution (L).
For 5 gels, prepare 500 ml of solution in a graduated cylinder, as follows:
25 ml 20 x concentrated Termination Solution (L)
475 ml Distilled or deionized water.
Mix well. Each group will require 50 ml of diluted Termination Solution.
4. On the day of the lab, set the incubation oven(s) at 37 C, 65 C,

and 80C.
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Experiment Results and Analysis
Actual results will yield broader bands of varying intensities. The
idealized schematic shows the relative positions of the bands, but the
results are not depicted to scale.
The largest fragments will migrate the slowest, the smallest will
migrate the fastest.
Option 1: Gel with 2 rows, 6 wells per row:
First Row
Lane Tube
2 B Mother DNA cut with Enzyme 1
3 C Mother DNA cut with Enzyme 2
4 D Child DNA cut with Enzyme 1
5 E Child DNA cut with Enzyme 2
Second Row
Lane Tube
2 F Father 1 DNA cut with Enzyme 1
3 G Father 1 DNA cut with Enzyme 2
4 H Father 2 DNA cut with Enzyme 1
5 I Father 2 DNA cut with Enzyme 2
( + )
( - )
1 2 3 4 5 6
23130 bp
9416 bp
6557 bp
4361 bp
3000 bp
2322 bp
2027 bp
725 bp
570 bp
23130 bp
9416 bp
6557 bp
4361 bp
3000 bp
2322 bp
2027 bp
725 bp
570 bp
Please refer to the kit
insert for the Answers to
Study Questions
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Appendices

A Agarose Gel Preparation For Southern Blot Analysis
B Quantity Preparations for Agarose Gel Electrophoresis

C Staining and Visualization of DNA with
InstaStain Ethidium Bromide Cards
Material Safety Data Sheets
Appendices
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Appendix
* 0.77 UltraSpec-Agarose gel percentage rounded up to 0.8%
0.8% Agarose Gel Electrophoresis Reference Tables
Time and Voltage recommendations for EDVOTEK
equipment are outlined in Table C.1 for 0.8%
agarose gels. The time for electrophoresis will
vary from approximately 15 minutes to 2 hours
depending upon various factors. Conduct the
electrophoresis for the length of time determined
by your instructor.
A
Amt of
Agarose
(g)
Concentrated
Buffer (50x)
(ml)
Size of Gel
(cm)
Distilled
Water
(ml)
Total
Volume
(ml)
7 x 7
7 x 10
7 x 14
0.23
0.39
0.46
0.6
1.0
1.2
29.4
49.0
58.8
+
= +
Individual 0.8%* UltraSpec-Agarose Gel
Table
A.1
30
50
60
Table
A.2
Amt of
Agarose
(g)
Diluted
Buffer (1x)
(ml)
Size of Gel
(cm)
7 x 7
7 x 10
7 x 14
0.23
0.39
0.46
30
50
60
+
Individual 0.8%*
UltraSpec-Agarose Gel
For DNA analysis, the recommended
electrophoresis buffer is Tris-acetate-
EDTA, pH 7.8. The formula for
diluting EDVOTEK (50x) concentrated
buffer is one volume of buffer concen-
trate to every 49 volumes of distilled
or deionized water. Prepare buffer as
required for your electrophoresis unit.
50x Conc.
Buffer (ml)
Distilled
Water (ml)
+
EDVOTEK
Model #
Total Volume
Required (ml)
Electrophoresis (Chamber) Buffer
M6+
M12
M36
300
400
1000
Dilution
Table
B
6
8
20
294
392
980
Time and Voltage Guidelines
(0.8% Gel)
Minimum / Maximum Volts
150
125
70
50
15 / 20 min
20 / 30 min
35 / 45 min
50 / 80 min
Table
C.1
EDVOTEK Electrophoresis Model
M6+ M12 & M36
Minimum / Maximum
25 / 35 min
35 / 45 min
60 / 90 min
95 / 130 min
If preparing a 0.8% gel with
concentrated (50x) buffer, use Table A.1
If preparing a 0.8% gel with
diluted (1x) buffer, use Table A.2
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Appendix
Agarose Gel Preparation - Quantity Preparations
B
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.
Bulk Electrophoresis Buffer
Quantity (bulk) preparation for 3 liters of 1x electrophoresis
buffer is outlined in Table D.
Batch Agarose Gels (0.8%)
For quantity (batch) preparation of 0.8% agarose gels, see
Table E.1.
1. Use a 500 ml ask to prepare the diluted gel buffer
2. Pour 3.0 grams 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 ask.
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 60C with swirl-
ing 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 ask in step 3.
6. Dispense the required volume of cooled aga-
rose solution for casting each gel. The volume required is
dependent upon the size of the gel bed and DNA staining
method which will be used. Refer to Appendix A or B for
guidelines.
7. Allow the gel to completely solidify. It will become rm
and cool to the touch after approximately 20 minutes.
Then proceed with preparing the gel for electrophoresis.
60C
Note: The UltraSpec-Agarose kit component is
often labeled with the amount it contains. In many
cases, the entire contents of the bottle is 3.0 grams.
Please read the label carefully. If the amount of
agarose is not specied or if the bottle's plastic seal
has been broken, weigh the agarose to ensure you
are using the correct amount.
Concentrated
Buffer (50x)
(ml)
Distilled
Water
(ml)
Total
Volume
(ml)
60 2,940 3000 (3 L)
= +
Bulk Preparation of
Electrophoresis Buffer
Table
D
3.0 7.5 382.5 390

Batch Preparation of
0.8% UltraSpec-Agarose
Amt of
Agarose
(g)
Concentrated
Buffer (50X)
(ml)
+
Distilled
Water
(ml)
Total
Volume
(ml)
= +
Table
E.1
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Appendix
C
Do not stain gel(s) in the electrophoresis chamber.
1. After electrophoresis, place the gel on a piece of plastic
wrap on a at surface. Moisten the gel with a few
drops of electrophoresis buffer.
2. Wearing gloves, remove and discard the clear plastic
protective sheet, and place the unprinted side of the
InstaStain Ethidium Bromide card(s) on the gel.
3. With a gloved hand, rmly run your ngers over the entire surface of the In-
staStain Ethidium Bromide card. Do this several times.
4. Place the gel casting tray and a small empty beaker on top to ensure that the
InstaStain Ethidium Bromide card maintains direct contact with the gel surface.
Allow the InstaStain Ethidium Bromide card to stain the gel for 3-5 minutes.
Note: Staining time is optimized for 0.8-1.0% gels. Gels of higher concentrations
will take longer to stain.
5. After 3-5 minutes, remove the InstaStain Ethidium Bromide card. Transfer the
gel to a ultraviolet (300 nm) transilluminator for viewing. Be sure to wear UV
protective goggles.
Caution: Ethidium Bromide is a listed mutagen.
Disposal of InstaStain Ethidium Bromide:
Disposal of InstaStain Ethidium Bromide cards and gels should follow institutional
guidelines for solid chemical waste.
Additional Notes About Staining:
If bands appear faint, or if you are not using EDVOTEK UltraSpec- Agarose, gels
may take longer to stain with InstaStain Ethidium Bromide. Repeat staining
and increase the staining time for an additional 3-5 minutes.
DNA markers should be visible after staining even if other DNA samples are faint
or absent. If markers are not visible, troubleshoot for problems with the electro-
phoretic separation.
Staining and Visualization of DNA
InstaStain Ethidium Bromide Cards
Wear Gloves
and Goggles
DNA InstaStain
Patents Pending DNA InstaStain
Patents Pending
DNA InstaStain
Patents Pending
DNA InstaStain
Patents Pending
-
-
-
-
-
DNA InstaStain
Patents Pending DNA InstaStain
Patents Pending
-
-
-
-
-
1
2
3
4
5
Press firmly.
Moisten
the gel.
Place the InstaStain
card on the gel.
Place a small weight to
ensure good contact.
View on U.V. (300 nm)
transilluminator
35
311
Experiment
Full-size (8.5 x 11) pdf copy of MSDS is available at www. edvotek.com or by request.
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2
Full-size (8.5 x 11) pdf copy of MSDS is available at www. edvotek.com or by request.
311
Experiment
36
S
t
a
b
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y
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c
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Electrophoresis can be completed

C. If the temperature exceeds 65

C incubation oven for 1 hour.

C for color development which begins with step 18.

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