Using Mitochondrial DNA
Polymorphisms in Evolutionary
and Forensic Biology Using Mitochondrial DNA Polymorphisms in
Evolutionary and Forensic Biology
IMPORTANT INFORMATION
Storage: Upon receipt of the kit, store proteinase K, mt primer/loading dye mix, and DNA marker
pBR322/BstNI in a freezer (approximately –20°C). All other materials may be stored at room temperature
(approximately 25°C).
Use and Lab Safety: The materials supplied are for use with the method described in this kit only. Use of
this kit presumes and requires prior knowledge of basic methods of gel electrophoresis and staining of DNA.
Individuals should use this kit only in accordance with prudent laboratory safety precautions and under the
supervision of a person familiar with such precautions. Use of this kit by unsupervised or improperly
supervised individuals could result in injury.
Limited License: Polymerase chain reaction (PCR) is protected by patents owned by Hoffman-La Roche,
Inc. The purchase price of this product includes a limited, non-transferable license under U.S. Patents
4,683,202; 4,683,195; and 4,965,188 or their foreign counterparts, owned by Hoffmann-La Roche Inc. and F.
Hoffmann-La Roche Ltd. (Roche), to use only this amount of the product to practice the Polymerase Chain
Reaction (PCR) and related processes described in said patents solely for the research, educational, and
training activities of the purchaser when this product is used either manually or in conjunction with an
authorized thermal cycler. No right to perform or offer commercial services of any kind using PCR,
including without limitation reporting the results of purchaser’s activities for a fee or other commercial
consideration, is hereby granted by implication or estoppel. Further information on purchasing licenses to
practice the PCR process may be obtained by contacting the Director of Licensing at The Perkin-Elmer
Corporation, 850 Lincoln Center Drive, Foster City, California 94404 or at Roche Molecular Systems, Inc.,
1145 Atlantic Avenue, Alameda, California 94501.
Printed material: The student instructions, pages 5-23, as well as the CarolinaBLU™ staining protocol on
page 29 may be photocopied as needed for use by your students. DNA
KITS
Learning Center Copyright © 2008, Dolan DNA Learning Center, Cold Spring Harbor Laboratory. All rights reserved. REAGENTS, SUPPLIES, AND EQUIPMENT CHECKLIST
Included in the kit: Needed but not supplied: DNA extraction and amplification (all kits):
£ 1.5 g Chelex® resin
£ 5 mL proteinase K (100 μg/mL)
£ 700 μL mt primer/loading dye mix
£ 25 *Ready-to-Go™ PCR Beads
£ 5 mL mineral oil
£ 130-μL tube pBR322/BstNI markers
(0.075 μg/μL)
£ Instructor’s manual with reproducible
Student Lab Instructions
£ MtDNA CD-ROM £ **Electrophoresis kits with ethidium bromide staining
(Kits 21-1237and 21-21-1237A) also include:
£ 5 g agarose
£ 150 mL 20∞ TBE
£ 250 mL ethidium bromide, 1 μg/mL
£ 4 latex gloves
£ 6 staining trays
**Electrophoresis kits with CarolinaBLU™ staining
(Kits 21-1238 and 21-1238A) also include:
£ 5 g agarose
£ 150 mL 20∞ TBE
£ 7 mL CarolinaBLU™ Gel & Buffer Stain
£ 250 mL CarolinaBLU™ Final Stain
£ 4 latex gloves
£ 6 staining trays £
£
£
£
£
£ £
£
£
£
£
£
£
£
£
£
£ 0.9% saline solution (NaCl), 10 mL per
student
in 15 mL tube
Micropipets and tips (1 μL to 1000 μL)
1.5 mL microcentrifuge tubes, polypropylene,
2 per student
Microcentrifuge tube racks
Microcentrifuge for 1.5-mL tubes
0.2 mL or 0.5 mL PCR tubes, 1 per student
(1.5 mL microcentrifuge tubes may also be used.)
0.2 mL or 0.5 mL tube adapters for
microcentrifuge (can be made from 0.5 mL
and/or 1.5 mL tubes)
Thermal cycler, programmable
Electrophoresis chambers
Electrophoresis power supplies
Gel staining trays
UV transilluminator (ethidium bromide
staining)
White light box (CarolinaBLU™ staining,
optional)
Camera or photo-documentary system
(optional)
Paper cup, 1 per student
Permanent markers
Container with cracked or crushed ice
Boiling water bath (optional, see instructions) *Ready-to-Go™ PCR Beads incorporate Taq
polymerase, dNTPs, and MgCl2. Each bead is
supplied in an individual 0.5–mL tube or a
0.2–mL tube.
**Electrophoresis reagents must be purchased
separately for Kits 21-1230 and 21-1230A. DNA
KITS
Learning Center Copyright © 2008, Dolan DNA Learning Center, Cold Spring Harbor Laboratory. All rights reserved. Using Mitochondrial DNA Polymorphisms in
Evolutionary and Forensic Biology
CONTENTS
STUDENT LAB INSTRUCTIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5
INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5 LAB FLOW . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7 METHODS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8 BIOINFORMATICS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .13 RESULTS AND DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .17 INFORMATION FOR INSTRUCTOR
CONCEPTS AND METHODS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .25 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .25 INSTRUCTOR PLANNING, PREPARATION, AND LAB FINE POINTS
CarolinaBLU™ STAINING
BIOINFORMATICS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .26 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .32 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .33 ANSWERS TO BIOINFORMATICS QUESTIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .33
ANSWERS TO DISCUSSION QUESTIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .34
CD-ROM CONTENTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .35 DNA
KITS
Learning Center Copyright © 2008, Dolan DNA Learning Center, Cold Spring Harbor Laboratory. All rights reserved. 5 STUDENT LAB INSTRUCTIONS
INTRODUCTION
In addition to the 46 chromosomes found in the nucleus of human cells,
each mitochondrion in the cell cytoplasm has several copies of its own
genome. The mitochondrial (mt) genome contains only 37 genes, all of
which are involved in the process of oxidative phosphorylation—the
production of energy and its storage in ATP.
There is strong evidence that the mitochondrion once existed as a freeliving bacterium that was taken up by a primitive ancestor of eukaryotic
cells. In this endosymbiosis, the host cell provided a ready source of
energy-rich nutrients, and the mitochondrion provided a means to
extract energy using oxygen. This attribute was key to survival, as oxygen
accumulated in the primitive atmosphere. Mitochondria are physically in
the same size range as bacteria, and the mt genome retains several
bacterial features. Like bacterial chromosomes and plasmids, the mt
genome is a circular molecule, and mt genes are not interrupted by
introns. These features are contrary to those of eukaryotic
chromosomes—which are linear—and eukaryotic genes—which have
numerous introns.
The entire DNA sequence of the human mt genome (16,569 nucleotides)
was determined in 1981, well in advance of the Human Genome Project.
Genes take up the majority of the mt genome. However, a noncoding
region of approximately 1200 nucleotides contains signals that control
replication of the chromosome and transcription of the mt genes. The
DNA sequence of the control region is termed “hypervariable,” because it
accumulates point mutations at approximately 10 times the rate of
nuclear DNA. This high mutation rate results in distinctive patterns of
single nucleotide polymorphisms (SNPs).
The combination of SNPs inherited by each person is termed a haplotype
(or “half type”). The mt genome provides only a half set of genes, because
it is inherited exlcusively from the mother with no paternal contribution.
The female egg is a huge cell with on the order of 100,000 mitochondria,
while the tiny sperm cell is powered by fewer than 100 mitochondria at
the base of the flagellum. Any male mitochondria that may enter the egg
cell at conception are identifed by their ubiquitin surface proteins as
“foreign” and actively destroyed by enzymes in the egg cytoplasm.
In the 1980s, Allan Wilson and coworkers at the University of California at
Berkeley used mtDNA polymorphisms to create a “family tree” showing
ancestral relationships between modern populations. Reasoning that all
human populations arose from a common ancestor in the distant
evolutionary past, Wilson’s group calculated how long it would take to
accumulate the pattern of mt mutations observed in modern
populations. They concluded that the ancestor of all modern humans
arose in Africa about 200,000 years ago.This common ancestor was widely
reported as the “mitochondrial Eve.”
While each cell contains only two copies of a given nuclear DNA sequence
— one on each of the paired chromosomes —there are hundreds to
Copyright © 2006, Dolan DNA Learning Center, Cold Spring Harbor Laboratory. All rights reserved. 6 Using Mitochondrial DNA Polymorphisms in Evolutionary and Forensic Biology thousands of copies of a given mt DNA sequence in each cell. This
amplification increases the chances that enough mtDNA can be obtained
for forensic analysis when tissue samlples are old or badly degraded. For
example, polymorphisms in the mt hypervariable region have been used
to:
• Identify remains from wars and natural disasters.
• Identify the remains of the Romanov royal family assasinated during
the Russian Revolution.
• Determine the relationship of Otzi, the Tyrolean ice man and the
ancient hominid Neandertal to modern humans.
Because of the high copy number of the mt genome, it offers the simplest
means for a person to visualize a discrete region of their own genetic
material. This experiment examines a sequence within the hypervariable
region of the mt genome. A sample of human cells is obtained by saline
mouthwash (alternatively DNA may be isolated from hair sheaths). DNA is
extracted by boiling with Chelex® resin, which binds contaminating metal
ions, and the control region sequence is amplified by polymerase chain
reaction (PCR).
However, since SNP polymorphisms do not change the size of the PCR
product (amplicon), gel electrophoresis shows no differences between
student samples. To analyze SNPs that vary from person to person, the
nucleotide sequence of each student amplicon must be determined. This
can be done by submitting student amplicons to the Sequencing Service
of the Dolan DNA Learning Center (DNALC) of Cold Spring Habor, which
will generate student mtDNA sequences and post the results via Internet
free of charge. Tools for generating mt haplotypes, comparing student
sequences, and studying human evolution comparing are found at the
DNALC’s BioServers Internet site (www.bioservers.org).
Biesecker L.G., Bailey-Wilson J.E., Ballantyne J., Baum H., Bieber F.R., Brenner C., Budowle B.,
Butler J.M., Carmody G., Conneally P.M., Duceman B., Eisenberg A., Forman L, Kidd
K.K., Leclair B., Niezgoda S., Parsons T.J., Pugh E., Shaler R., Sherry S.T., Sozer A. and
Walsh A. (2005. Epidemiology. DNA Identifications After the 9/11 World Trade Center
Attack. Science 310(5751):1122-1123. Cann R.L., Stoneking, M. and Wilson, A.C. (1987).
Allan C. Mitochondrial DNA and Human Evolution. Nature 325: 31-36.
Gill, P., Iavanov, P.L., Kimpton, C., Piercy, R., Benson, N., Tully, G., Evett, I., Haqelberg, E. and
Sullivan, K. (1994). Identification of the Remains of the Romanov Family by DNA
Anaylsis. Nature Genetics 6(2): 130-135.
Handt O., Richards M., Trommsdorff M., Kilger C., Simanainen J., Georgiev O., Bauer K.,
Stone A., Hedges R., Schaffner W, et al. (1994). Molecular genetic analyses of the
Tyrolean Ice Man. Science 264(5166):1775-1778.
Krings M., Stone A., Schmitz R.W., Krainitzki H., Stoneking M. andPääbo S. (1997).
Neandertal DNA Sequences and the Origin of Modern Humans. Cell 90(1):19-30.
Mullis, K. (1990). The Unusual Origin of the Polymerase Chain Reaction. Scientific American
262(4): 56-65. DNA
KITS
Learning Center Copyright © 2008, Dolan DNA Learning Center, Cold Spring Harbor Laboratory. All rights reserved. Using Mitochondrial DNA Polymorphisms in Evolutionary and Forensic Biology 7 LAB FLOW
I. ISOLATE DNA FROM CHEEK CELLS 99°C (ALTERNATE) I. ISOLATE DNA FROM HAIR SHEATHS 37°C 99°C II. AMPLIFY DNA BY PCR III. ANALYZE PCR PRODUCTS BY GEL ELECTROPHORESIS Copyright © 2008, Dolan DNA Learning Center, Cold Spring Harbor Laboratory. All rights reserved. Using Mitochondrial DNA Polymorphisms in Evolutionary and Forensic Biology 8 METHODS I. ISOLATE DNA FROM CHEEK CELLS Reagents Supplies and Equipment 0.9% Saline solution, 10 mL
10% Chelex®, 100 μL (in 0.2 or 0.5 mL PCR
tube) Permanent marker
Paper cup
Micropipets and tips (10-1000 μL)
1.5 mL microcentrifuge tubes
Microcentrifuge tube rack
Microcentrifuge adapters
Microcentrifuge
Thermal cycler (or water bath or heat
block)
Container with cracked or crushed ice
Vortexer (optional) 1. Use a permanent marker to label a 1.5 mL tube and paper cup with
your assigned number.
2. Pour saline solution into your mouth, and vigorously rinse your cheek
pockets for 30 seconds.
3. Expel saline solution into the paper cup.
4. Swirl cup gently to mix cells that may have settled to the bottom. Use
micropipet with fresh tip to transfer 1500 μL of the solution into your
labeled 1.5-mL microcentrifuge tube.
5. Place your sample tube, along with other student samples, in a
balanced configuration in a microcentrifuge, and spin for 90 seconds
at full speed.
Before pouring off supernatant,
check to see that pellet is firmly
attached to tube. If pellet is loose
or unconsolidated, carefully use
micropipet to remove as much
saline solution as possible. 6. Carefully pour off supernatant into the paper cup. Try to remove most
of the supernatant, but be careful not to disturb cell pellet at the
bottom of the tube. (The remaining volume will approximately reach
the 0.1 mark of a graduated tube.) Food particles will not resuspend. 7. Set micropipet to 30 μL. Resuspend cells in the remaining saline by
pipetting in and out. Work carefully to minimize bubbles. Alternatively, you may add the cell
suspension to Chelex in a 1.5 mL
tube, and incubate in a boiling
water bath or heat block. 8. Withdraw 30 μL of cell suspension, and add to a PCR tube
containing 100 μL of Chelex®. Label the cap and side of the tube
with your assigned number. Your teacher may instruct you to
collect a sample of cell suspension to
observe under a microscope. 9. Place your PCR tube, along with other student samples, in a thermal
cycler that has been programmed for one cycle of the following
profile. The profile may be linked to a 4°C hold program.
Boiling step: The near-boiling temperature lyses
the cell and nuclear membranes,
releasing DNA and other cell
contents. DNA
KITS
Learning Center 99°C 10 minutes 10. After boiling, vigorously shake the PCR tube for 5 seconds. Copyright © 2008, Dolan DNA Learning Center, Cold Spring Harbor Laboratory. All rights reserved. Using Mitochondrial DNA Polymorphisms in Evolutionary and Forensic Biology To use adapters, “nest” the sample
tube within sequentially larger
tubes: 0.2 mL within 0.5 mL within
1.5 mL. Remove caps from tubes
used as adapters. 9 11. Place your tube, along with other student samples, in a balanced
configuration in a microcentrifuge, and spin for 90 seconds at full
speed. If your sample is in a PCR tube, one or two adapters will be
needed to spin the tube in a microcentrifuge designed for 1.5 mL tubes.
12. Use a micropipet with fresh tip to transfer 30 μL of the clear
supernatant into a clean 1.5 mL tube. Be careful to avoid pipetting
any cell debris and Chelex® beads.
13. Label the cap and side of the tube with your assigned number. This
sample will be used for setting up one or more PCR reactions.
14. Store your sample on ice or at -20°C until you are ready to continue
with Part II.
I. (ALTERNATE) ISOLATE DNA FROM HAIR SHEATHS Your teacher may instruct you to
prepare a hair sheath to observe
under a microscope. HAIR WITH
SHEATH HAIR
ROOT BROKEN
HAIR Reagent Supplies and Equipment 100 mg/mL proteinase K, 100 μL (in 0.2 or
0.5 mL tube) Permanent marker
Scalpel or razor blade
Forceps or tweezers
Thermal cycler (or water bath or heat
block)
Container with cracked or crushed ice
Vortexer (optional) 1. Pull out several hairs and inspect for presence of a sheath. The sheath
is a barrel-shaped structure surrounding the base of the hair, and can
be readily observed with a hand lens or dissecting microscope. The
glistening sheath can be observed with the naked eyes by holding
the hair up to a light source. (Sheaths are most easily observed on
dark hair.)
2. Select one to several hairs with good sheaths. Alternately, select hairs
with the largest roots. Broken hairs, without roots or sheaths, will not
yield enough DNA for amplification.
3. Use a fresh razor blade or scalpel to cut off hair shafts just above the
sheath.
4. Use forceps to transfer hairs to a PCR tube containing 100 μL of
proteinase K. Make sure sheath is submerged in the solution and not
stuck on the test tube wall. Label the cap and side of the tube with
your assigned number. Alternatively, you may add the
hairs to proteinase K in a 1.5 mL
tube, and incubate in a water bath
or heat block. 5. Place your PCR tube, along with other student samples, in a thermal
cycler that has been programmed for one cycle of the following
profile.
Incubation Step: 37°C 10 minutes 6. Remove sample tube to room temperature. Vortex by machine or
vigorously with finger for 15 seconds to dislodge cells from hair shaft. Copyright © 2008, Dolan DNA Learning Center, Cold Spring Harbor Laboratory. All rights reserved. 10 Using Mitochondrial DNA Polymorphisms in Evolutionary and Forensic Biology 7. Place your PCR tube, along with other student samples, in a thermal
cycler that has been programmed for one cycle of the following
profile. The profile may be linked to a 4°C hold program.
Boiling step: 99°C 10 minutes 7. Remove sample tube to room temperature, and mix by pipetting in
and out for 15 seconds.
9. Store your sample on ice or in the freezer until ready to begin Part II. II. AMPLIFY DNA BY PCR
Reagents (at each student station) Supplies and Equipment *Cheek cell DNA. 2.5 μL (from Part I)
*Mt primer/loading dye mix, 25 μL
Ready-To-GoTM PCR beads (in 0.2 mL or
0.5 mL PCR tube) Permanent marker
Micropipet and tips (1-100 μL)
Microcentrifuge tube rack
Thermal cycler
Container with cracked or crushed ice Shared Reagent
Mineral oil, 5 mL (depending on thermal
cycler)
*Store on ice 1. Obtain a PCR tube containing a Ready-To-Go™ PCR Bead. Label with
your assigned number.
The primer/loading dye mix will turn
purple as the PCR bead dissolves. If the reagents become splattered
on the wall of the tube, pool them
by pulsing in a microcentrifuge or
by sharply tapping the tube
bottom on the lab bench.
If your thermal cycler does not
have a heated lid: Prior to thermal
cycling, you must add a drop of
mineral oil on top of your PCR
reaction. Be careful not to touch
the dropper tip to the tube or
reaction, or the oil will be
contaminated with your sample. DNA
KITS
Learning Center 2. Use a micropipet with fresh tip to add 22.5 μL of mt primer/loading
dye mix to the tube. Allow the bead to dissolve for a minute or so.
3. Use a micropipet with fresh tip to add 2.5 μL of your cheek cell DNA
(from Part I) directly into the primer/loading dye mix. Insure that no
cheek cell DNA remains in the tip after pipetting.
4. Store your sample on ice until your class is ready to begin thermal cycling.
5. Place your PCR tube, along with other student samples, in a thermal
cycler that has been programmed for 30 cycles of the following
profile. The profile may be linked to a 4°C hold program after the 30
cycles are completed.
Denaturing step:
Annealing step:
Extending step: 94°C
58°C
72°C 30 seconds
30 seconds
30 seconds 6. After cycling, store the amplified DNA on ice or at -20°C until you are
ready to continue with Part III. Copyright © 2008, Dolan DNA Learning Center, Cold Spring Harbor Laboratory. All rights reserved. Using Mitochondrial DNA Polymorphisms in Evolutionary and Forensic Biology 11 III. ANALYZE PCR PRODUCTS BY GEL ELECTROPHORESIS
Reagents Supplies and Equipment *PCR product (from Part II), 15 μL Micropipet and tips (1-100 μL)
Microcentrifuge tube rack
Gel electrophoresis chamber
Power supply
Staining trays
Latex gloves
UV transilluminator (for use with ethidium
bromide)
White light transilluminator (for use with
CarolinaBLU™)
Digital or instant camera (optional)
Water bath (60°C)
Container with cracked or crushed ice Shared Reagents
*pBR322/BstNI marker
1.5% agarose in 1X TBE, 50 mL
1X TBE, 300 mL
Ethidium bromide (1 μg/mL), 250 mL
or
CarolinaBLU™ Gel & Buffer Stain, 7 mL
CarolinaBLU™ Final Stain, 250 mL
*Store on ice 1. Seal the ends of the gel-casting tray with masking tape, and insert a
well-forming comb.
Avoid pouring an overly thick gel,
which is more difficult to visualize.
The gel will become cloudy as it
solidifies. 2. Pour 1.5% agarose solution to a depth that covers about 1/3 the
height of the open teeth of the comb.
3. Allow the gel to solidify completely. This takes approximately 20
minutes. Do not add more buffer than
necessary. Too much buffer above
the gel channels electrical current
over the gel, increasing running
time. 4. Place the gel into the electrophoresis chamber, and add enough 1X
TBE buffer to cover the surface of the gel. 100-bp ladder may also be used as
a marker. 6. Use a micropipet with a fresh tip to load 20 μL of pBR322/BstNI size
marker into the far left lane of the gel. Expel any air from the tip before
loading. Be careful not to push the
tip of the pipet through the
bottom of the sample well. 7. CAUTION: DO NOT LOAD YOUR ENTIRE SAMPLE! Use a micropipet
with a fresh tip to add 15 μL of your sample/loading dye mixture into
your assigned lane of a 1.5% agarose gel, according to the diagram
below. (If you used mineral oil during PCR, pierce your pipet tip
through the layer of mineral oil to withdraw the PCR sample and 5. Carefully remove the comb, and add additional 1X TBE buffer to just
cover and fill in wells – creating a smooth buffer surface. MARKER
pBR322/
BstNI –� + 1 2 STUDENT SAMPLES
3
4 5 6 leave the mineral oil behind in the original tube.)
8. Store the remaining 10 μL of your mt PCR product on ice for
subsequent use in DNA sequencing. Copyright © 2008, Dolan DNA Learning Center, Cold Spring Harbor Laboratory. All rights reserved. 12 Using Mitochondrial DNA Polymorphisms in Evolutionary and Forensic Biology 9. Run the gel at 130 V for approximately 30 minutes. Adequate
separation will have occurred when the cresol red dye front has
moved at least 50 mm from the wells.
10. Stain the gel using ethidium bromide or CarolinaBLU™:
Destaining the gel for 5-10
minutes in tap water leaches
unbound ethidium bromide from
the gel, decreasing background
and increasing contrast of the
stained DNA. a. For ethidium bromide, stain 10-15 minutes. Decant stain back into
storage container for reuse, and rinse gel in tap water. Use gloves
when handling ethidium bromide solution and stained gels or
anything that has ethidium bromide on it. Ethidium bromide is a
known mutagen and care should be taken when using and disposing
of it.
b. For CarolinaBLU™, follow directions in the Instructor Planning
section. Transillumination, where the light
source is below the gel, incr...

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