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Cloning and Sequencing Explorer Series
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Why Teach
Cloning and
Sequencing
Series?
• Students guide the research process and
make decisions about their next steps
• Encompasses a myriad of laboratory skills
and techniques commonly used in research
• Students generate original data that may
lead to publications in GenBank
• Students formulate scientific explanations
using data, logic, and evidence
• Students understand research is a process
rather than a single experiment giving
students a real-life research experience
with both its successes and challenges
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Appropriate
courses
• Molecular Biology
• Recombinant DNA Techniques
• Biotechnology
• Molecular Evolution
• Bioinformatics
• Advanced Cell Biology
• Advanced Genetics
• Advanced Plant Biology
• Independent Research
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Laboratory
Overview
Genomic DNA Extraction
DNA Precipitation
DNA Quantitation
Bioinformatics
Sequence Data Editing
Contig Assembly
Intron-Exon Prediction
GAPDH PCR
Nested PCR
Degenerate primers
Exonuclease
Sequencing
Automated sequencing
Gel Electrophoresis
DNA Gel Interpretation
Band Identification
Standard Curve Use
Plasmid Miniprep
Restriction Enzyme
Digestion
Gel Electrophoresis
PCR Purification
Size Exclusion Chromatography
Microbial Culturing
Antibiotic Selection
Sterile Technique
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Cloning
Direct PCR cloning
Transformation
Ligation
Student use the
following
techniques
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Micropipetting
DNA extraction
Gel electrophoresis & interpretation
Polymerase chain reaction
DNA purification
Restriction enzyme digests
Microbiological sterile technique
Preparing competent bacteria
DNA ligation
Heat-shock transformation
Plasmid DNA isolation
Sequence analysis
BLAST searching
GenBank submission
Day 1
Day 2
Chapter
Task
1: Nucleic Acid Extraction
Extract DNA
2: GAPDH PCR
Initial PCR, Exo, Nested PCR
3: Electrophoresis
Electrophorese PCR products
4: PCR Purification
Purify PCR Products
5: Ligation
Ligate PCR Product
6: Transformation
Transformation
7: Plasmid Purification
Mini-prep and restriction
digest confirmation
8: Sequencing
Prep and send sequencing
reactions
Analyze Sample Sequence
9: Bioinformatics
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DNA Preparation
and PCR
Amplification of
Genomic DNA Extraction
DNA Precipitation
DNA Quantitation
GAPDH
GAPDH PCR
Nested PCR
Degenerate primers
Exonuclease
Gel Electrophoresis
DNA Gel Interpretation
Band Identification
Standard Curve Use
• Students choose plant tissue
• Two rounds of PCR
– Round 1: Use of degenerate primers
– Round 2: Nested PCR
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Chapter 1
1: Nucleic Acid
Extraction
Optional: Dependent on time constraints, analyze
samples prior to the next steps. This may include
agarose gel electrophoresis, fluorometry, or
spectrometry.
Optional: If time permits proceed directly to Step 1–2
and set up PCR reactions using freshly extracted
genomic DNA.
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DNA Extraction
• Use young, fresh plant-tissue
• DNA extraction at room temperature
• Time requirement ~30 minutes
• Does not require DNA quantification
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Benefits of
using plants
• Large number of species
• Lots of diversity
• Phylogenetic approaches
• Avoid ethical concerns associated
with animals
• No pre-approval
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Nucleic Acid
Extraction
Quick Guide
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Chapter 2
2: GAPDH PCR
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Optional: Although this protocol recommends analyzing the PCR products after both rounds of
PCR have been completed, PCR results can be assessed using electrophoresis directly after
this reaction is complete. Positive controls should yield visible bands. It is possible that some
plant genomic DNA will not yield a visible band during the initial round of PCR and yet still be
amplified after the second round of nested PCR. Note: If this is done — DO NOT add loading
dye directly to the PCR reactions as loading dye may interfere with the subsequent round of
PCR.
PCR Reactions
Initial
Nested
• Color-coded PCR primers
(hallmark of Bio-Rad PCR kits)
• Two positive controls
• Arabidopsis
• pGAP (plasmid DNA)
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• One negative control
What is a
Housekeeping
Gene?
Highly conserved genes that must be
continually expressed in all tissues of
organisms to maintain essential cellular
functions.
Examples:
•GAPDH
•Cytochrome C
•ATPase
•ß-actin
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Why use GAPDH?
Glyceraldehyde
3-Phosphate
Dehydrogenase
(GAPDH)
• Enzyme of glycolysis
• Structure and
reaction mechanism
well-studied
• Multitude of
sequences
• Highly conserved
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Gene
Families
gene
duplication
Endosymbiotic
event
GAPA
gene
duplication
GAPC/CP
GAPA/B
Host Cell
GAPC
Most algae
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Land
plants
Mesostigma
(small group
of green
algae)
Initial PCR may
result in some
amplicons that
are non-specific
Because of the
degenerate primer
used to amplify the
GAPDH of various
plant species the
initial PCR may
also result in some
non-specific
amplifications
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Nested PCR
amplifies only
regions within
the GAPDH
gene
Nested PCR is
more specific
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PCR Animation
http://www.bio-rad.com/flash/07-0335/07-0335_PCR.html
DNA sequence
varies between
species
Primers need to
be designed to
account for
species
variation
• What if you don’t know the exact DNA
target sequence?
• How do you design primers?
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Variation in
the Genetic
Code
The GAPDH
enzyme (protein)
is highly
conserved but
there are
variations at the
DNA level
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Introns are less conserved since they do
not code for protein
Conservative substitution-does not change protein
properties
Degenerate
primers are
used to
account for
sequence
variation
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Primers are
designed using
the consensus
sequence
Plant
GAPDH
Tobacco
Carrot
Blue gem
Tomato
Snapdragon
Accession
Number
DQ682459
AY491512
X78307
AB110609
X59517
Sequence
GATTTCGTTGTGGAATCCACTGG
GAGTACATTGTGGAGTCCACTGG
GAGTACGTCGTTGAGTCGACTGG
GACTTCGTTGTTGAATCAACCGG
GAGTATATTGTGGAGTCCACTGG
Consensus sequence GABTATGTTGTTGARTCTTCWGG
Primer set:
GA(GTC)TATGTTGTTGA(GA)TCTTC(AT)GG
Yield: 12 primers
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Reverse primers are designed in the same
fashion
Degenerate
primers have
optional bases
in specified
positions
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To increase the probability that the
primer will anneal to the target DNA,
variable bases are designed into the
primer.
Multiple oligos
comprise the
forward primer
GAGTATGTTGTTGA(GA)TCTTC(AT)GG
GATTATGTTGTTGA(GA)TCTTC(AT)GG
GACTATGTTGTTGA(GA)TCTTC(AT)GG
Position 3
has 3 bases
GA(GTC)TATGTTGTTGAGTCTTC(AT)GG Position 15
GA(GTC)TATGTTGTTGAATCTTC(AT)GG has 2 bases
GA(GTC)TATGTTGTTGA(GA)TCTTCAGG Position 21
GA(GTC)TATGTTGTTGA(GA)TCTTCTGG has 2 bases
3 x 2 x 2 = 12
different oligonucleotides comprising the
forward primer
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DNA Isolation
and
Amplification
To identify differences in GAPDH code we must
isolate plant DNA and amplify the gene of interest
using PCR first with degenerate primers (primers
that account for variation in the DNA code)
A second PCR reaction (Nested PCR) is necessary
to amplify the region which contains one of the
GAPDH gene sequences (second set of primers are
nested inside the initial PCR product sequence)
Biotechnology Explorer PCR primers are
color-coded
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Setting up
initial PCR
Reactions
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Setting up
Initial PCR
Reactions
Protocol
1. Add 20 µl of blue mastermix with
initial primers to each PCR tube
2. Add 15 µl of sterile water to each tube
3. Add 5 µl of DNA template to the
appropriate tube
•
Control Arabidopsis gDNA
•
Control plasmid DNA
•
Test gDNAs
•
Negative control
4. Amplify in thermal cycler (Annealing
temp 52oC)
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Results of Initial
PCR Reactions
1% agarose gel loaded
with (20 µl) initial PCR
samples.
Green bean and Lamb’s
ear gDNA samples
generated using Nucleic
Acid Extraction module.
1
2
3
4
5
2000 bp1500 bp1000 bp500 bp-
Lane 1- 500 bp molecular weight ruler (10 µl),
Lane 2- PCR of control Arabidopsis gDNA with initial primers (20 µl)
Lane 3- PCR of green bean gDNA with initial primers (20 µl)
Lane 4- PCR of lamb’s ear gDNA with initial primers (20 µl)
Lane 5- PCR of pGAP plasmid control with initial primers (20 µl)
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Sometimes no amplification is observed with initial PCR with
some plants, or much fainter than this gel
Why is a
Nested PCR
reaction
necessary?
• Use of degenerate primers may not give
you an exact match to the target sequence
INITIAL
FORWARD PRIMER
GACTATGTTGTTGAGTCTTCTGG
Arabidopsis
GAPC1AT3G04120
GACTACGTTGTTGAGTCTACTGG
• Because there are multiple primers in the
mix, the primer concentration for the
matching primer is lower than normal
(1/12th concentration)
• Problems with initial PCR:
– inefficient
– non-specific
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• Benefits of initial PCR:
– cast a wide net
– increase the pool of specific products
Exonuclease 1
treatment is
needed before
the second
round of PCR
(nested PCR) is
done
• The primers that were not incorporated into
PCR product in the first reaction must be
removed so that they do not amplify target
DNA in the second round of PCR.
• Exonuclease I will be added to the PCR
products
• The enzyme must be inactivated before
proceeding to the nested PCR
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Exo treatment
Protocol
1. Add 1 µl of exonuclease 1 enzyme to the
PCR reactions
2. Incubate 15 min 37°C
3. Incubate 15 min 80°C
4. Dilute 2 µl Exo-treated PCR product in
98 µl water
Note: Thermal cycler can be programmed
for exo incubations
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Nested PCR
amplifies only
regions within
the GAPDH
gene
Nested PCR is
more specific
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Setting up
Nested PCR
Reactions
Protocol
1. Mix 20 µl of diluted exo-treated
template DNA with 20 µl of
yellow mastermix with nested
primers
2. For controls, mix 20 µl of control
pGAP plasmid and 20 µl of water
with 20 µl of yellow mastermix
with nested primers
3. Amplify in thermal cycler
(Annealing temp 46oC)
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PCR Products
Initial vs. Nested
Reactions
GAPC
GAP-C2
GAPCP-1
GAPCP-2
Nested GAPDH PCR product
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Using Nested
PCR to
increase your
final PCR
product
Initial PCR
DNA template:
Genomic DNA
Nested PCR
DNA template:
Initial PCR products
• There is more PCR product from the nested
PCR reactions since there is more specific
template DNA to start from
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• Results: intense, bold band on agarose gel
Chapter 3
3: Electrophoresis
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Electrophoresis
Quick Guide
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PCR results
1
MW
2
Arabidopsis
I
1% agarose gel loaded
with 20 µl initial PCR
samples and 5 µl
nested PCR samples.
2000 bp1500 bp1000 bp500 bp-
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3
N
4
5
Green bean
I
N
6
7
8
Lamb’s ear
I
N
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pGAP
I
N
Laboratory
Overview
Genomic DNA Extraction
DNA Precipitation
DNA Quantitation
Bioinformatics
Sequence Data Editing
Contig Assembly
Intron-Exon Prediction
GAPDH PCR
Nested PCR
Degenerate primers
Exonuclease
Sequencing
Automated sequencing
Gel Electrophoresis
DNA Gel Interpretation
Band Identification
Standard Curve Use
Plasmid Miniprep
Restriction Enzyme
Digestion
Gel Electrophoresis
PCR Purification
Size Exclusion Chromatography
Microbial Culturing
Antibiotic Selection
Sterile Technique
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Cloning
Direct PCR cloning
Transformation
Ligation
Day 1
Day 2
Chapter
Task
1: Nucleic Acid Extraction
Extract DNA
2: GAPDH PCR
Initial PCR, Exo, Nested PCR
3: Electrophoresis
Electrophorese PCR products
4: PCR Purification
Purify PCR Products
5: Ligation
Ligate PCR Product
6: Transformation
Transformation
7: Plasmid Purification
Mini-prep and restriction
digest confirmation
8: Sequencing
Prep and send sequencing
reactions
Analyze Sample Sequence
9: Bioinformatics
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PCR Purification,
Ligation,
Transformation
and Plasmid
Miniprep
Purification
Plasmid Miniprep
Restriction Enzyme
Digestion
Gel Electrophoresis
PCR Purification
Size Exclusion Chromatography
Microbial Culturing
Antibiotic Selection
Sterile Technique
Cloning
Direct PCR cloning
Transformation
Ligation
• Choose best PCR products for ligation
• Transformation and selection
• Plasmid Miniprep preparation
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Chapter 4
4: PCR Purification
Additional tasks to perform prior to next stage:
Starter cultures must be inoculated one day prior to the
transformation with a starter colony from the HB101 LB
agar starter plate. Incubate cultures with shaking
overnight at 37oC.
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Optional: Electrophorese 5 µl of the purified sample along with 5 µl of
the unpurified sample on an agarose gel.
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Column
Purification of
PCR products
SEC is used to purify
large PCR products
from the smaller
primers, dNTPs and
enzymes.
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PCR Purification
Quick Guide
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Ligation and
Transformation
• Blunt-end PCR product & ligate to pJet
vector
• Preparation of competent bacteria cells
• Efficient heat-shock transformation
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Chapter 5
5: Ligation
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Ligation
Sticky End Ligation:
If the insert has sticky
ends then the vector
should be cut with the
same enzyme to produce
complementary ends
Allows for directional
cloning (cDNA)
Blunt End Ligation:
All DNA ends are
compatible, not necessary
to cut vector and insert
with the same restriction
enzymes
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Treating the PCR product
with a proofreading DNA
polymerase removes 3’-A
added by Taq DNA
polymerase in PCR,
leaving blunt ends ready
for ligation.
pJet1.2 Blunted
Vector
2974 bp in length
Designed for blunt-end
cloning
MCS (Multiple Cloning Site)
has restriction enzyme sites
may be used for later
manipulation
High copy number
b-lactamase gene for Amp
resistance
Contains the eco47IR gene
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Positive
Selection
The eco471R gene
codes for the Eco47I
restriction enzyme
which is toxic to E.coli.
When the eco471R gene is disrupted by the
insertion of DNA into the cloning site, the gene will
no longer be expressed and the transformed cells
will grow on selective media.
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Possible Ligation
Products
Expected Results:
Self-ligation of vector
Ligation of vector with
multiple inserts
Ligation of vector with
primer-dimers or short
DNA fragments
Self-ligation of insert
Ligation of one insert
into vector
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Ligation
Quick Guide
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Chapter 6
6: Transformation
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Transformation
Competent cells
pGAP plasmid control
IPTG
Competent Cells are very delicate: keep on ice
Heat Shock
Bacterial transformation with ligation mixtures is very
inefficient (less efficient than transformation with plasmid
DNA). Transform with pGAP plasmid as control
Isopropyl b-D-1-thiogalactopyranoside (IPTG) is added to
the selective medium to artificially increase the expression
of the ampr gene and increase transformation efficiency
Heat Shock: plating bacteria from ice to agar plates at 370C
Transformation
Quick Guide
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Chapter 7
7: Plasmid Purification
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Plasmid
Minipreps
• Isolate plasmid DNA
• Restriction digest
• Electrophorese to confirm inserts
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Picking
colonies for
plasmid
minipreps
65
Each colony is a
clonal growth
(clones)
from one
transformed
bacteria
Plasmid
Purification and
Restriction
Digest Analysis
Quick Guide
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Optional: It is recommended that 5 µl of undigested DNA also be run
next to your digested samples. Prepare these samples by combining
5 µl of miniprep DNA with 5 µl of sterile water.
Analysis of
plasmid digests
Bgl II Digest
GAPDH
inserts
2.5 kb >
2.0 kb >
1.5 kb >
1.0 kb >
0.5 kb >
pJet vector
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Example of a
miniprep
digestion with
Bgl II
1
2
3
4
5
6
7
8
9
pJet vector
GAPDH
inserts
Digested and
undigested DNA were
electrophoresed on a
1% TAE agarose gel
Lane 1: 500 bp molecular weight ruler
Lanes 2, 4, 6, 8: minipreps digested with BglII
Lanes 3, 5, 7, 9: undigested minipreps
• Different sizes of inserts suggests different
GAPDH genes were cloned in this ligation
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• Inserts can vary from 0.5–2.5 kb depending on
plant species
Chapter 8
8: Sequencing
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Sequencing
• Primers, control plasmid and barcoded 96well plate are included. Actual sequencing
service is not included with the purchase of
the series.
• Bio-Rad Laboratories has been working
with Eurofins MWG/Operon which is a
company that offers sequencing services
around the world. We have been able to
partner with Eurofins MWG/Operon so that
they can offer a highly discounted rate. The
benefit of being able to utilize the
discounted services of Eurofins
MWG/Operon is that their turnaround time is
1-2 days and as an ISO9001 company they
adhere to specific quality standards. To
contact Eurofins MWG/Operon please visit
www.operon.com/bio-rad or call (800)6882248.
• Alternate options for sequencing would
include local universities and other
professional sequencing services.
Sequencing
Methods
• Maxam-Gilbert: Chemical Degradation
– 5’-end of DNA labeled with a radioactive tag
– Labled DNA is divided into 4 test tubes with
chemicals which cleave after a particular base
– PAGE to seperate cleaved fragments
– X-ray film (autoradiography)
– Derive DNA sequence from X-ray film image
– Limited to about 100 bases
• Sanger: Chain Termination
– Single-stranded template of DNA
– Template DNA is divided into 4 test tubes with
• Sequencing primers
• DNA polymerase
• Labled nucleotides (dNTP’s)
• Modified nucleotide, different dideoxynucleotide (ddNTP)
to each reaction tube
– Allow for DNA synthesis, when ddNTP is
incorporated polymerisation will stop
– PAGE and autoradiography can be used to separate
and visualize fragment sizes and read sequence
Setting up
Sequencing
Reactions
• Add sequencing primers to DNA
• Load 96-well plate
• Send sealed plate off to chosen
sequencing facility for sequencing
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Setting up
Sequencing
Reactions
GAPDH gene of interest
Always need to
sequence
reverse,
complementary
strand
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pJet cloning vector
Why use
multiple
sequencing
primers?
Typical sequencing reactions yield 500-600
bases of sequence.
If the GAPDH insert is longer a single set of
sequencing primers will not lead to the full
sequence.
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Sequencing
• Automated Sanger method of sequencing
• 4 fluorescent dyes- 1 for each base
• DNA fragments separated by CE
• Fragments separated in sequential order
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Chapter 9
9: Bioinformatics
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Bioinformatics
• Two month subscription to genetic analysis
software from Geospiza
• Data is stored on iFinch server
• Data can be accessed 24/7
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