InfoRefsDataWorldMapMain.docx

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Premise
DNA Evidence is routinely used as a means to determine the
identity of a suspect that has left DNA at a crime scene.
If a suspect does not match the evidence left at a crime scene,
they can be excluded with near certainty.
If the suspect does share his/her genotype with the evidence, he
can not be excluded as the perpetrator.
The number of loci genotyped and the allele frequencies
represented determine the likelihood of a false positive match.

FREDONIA
Damn Footprints!!!
Backstory
A crime has been committed in the new Science Center. Over
the winter break a trespasser broke into the science center and
walked across the freshly poured floors in their dirty boots. As
a result the floors in the second floor hallway has been ruined
and must be replaced. University police arrived on the scene in
time to see a single person running from the worksite. While the
perpetrator was not apprehended, they did loose their hat on the
fence as they made their escape. Several hairs were found in the
hat with the follicles intact. The police have provided us with
DNA extracted from these follicles in hopes that we can
identify the perpetrator and bring them to justice. It is up to you
to solve this hairy crime and identify the person responsible for
delaying the completion of the Biology department's new home.
Premise
Failure to exclude a suspect is not the same as proving that
he/she is the perpetrator.
Confidence is assessed by considering several loci and
considering the allele frequency at each locus.

By using neutral polymorphisms (no selective pressure),
estimates can be made about the expected frequency of the
indicated alleles randomly occurring in the population.
Such loci conform to Hardy-Weinberg equilibrium.
Population Genetics
Population: A group that lives in the same geographic area and
can successfully reproduce.
Gene Pool: Genes present in a population.
Allele Frequency: For a specific gene, the relative frequency of
each allele. The sum of all allele frequencies must = 1.
Calculation of allelic frequencies in gametes
Hardy-Weinberg Equilibrium
p + q = 1
p = allele frequency of one allele
q = allele frequency of a second allele
p2 + 2pq + q2 = 1
p2 and q2 genotype frequencies for each homozygote
2pq genotype frequency for heterozygotes
All of the allele frequencies together equal 1
All of the genotype frequencies
together equal 1
Genotype Frequencies In Progeny
Paternal Gametes

A(p) a(q)
Maternal A(p) AA(p2) Aa(pq)
Gametes a(q) Aa(pq) aa(q2)
genotype: AA Aa aa
frequency: p2 2pq q2
Or Mathematically: (p+q)(p+q) = p2 + 2pq + q2 = 1
A Punnett square showing the ratio p2:2pq:q2
Implications of the Hardy-Weinberg Principle
Allelic frequencies remain constant from generation to
generation
p + q = 1
frequencies of AA, Aa, and aa genotypes
are p2, 2pq, and q2
frequency p' of allele A in the next generation
p' = p2 + 2pq/2 = p(p+q) = p
OffspringMating TypeFrequencyAAAaaaAA x AAp4p4AA x aa
(2 ways)2p2q22p2q2aa x aaq4q4AA x Aa (2
ways)4p3q2p3q2p3qAa x Aa4p2q2p2q22p2q2p2q2Aa x aa (2
ways)4pq32pq32pq3AA offspring = p4 + 2p3q + p2q2 = p2(p2 +
2pq + q2) = p2(p + q)2 = p2(1)2 = p2.Aa offspring = 2p3q +
4p2q2 + 2pq3 = 2pq(p2 +2pq + q2) = 2pq(p + q)2 = 2pq(1)2 =
2pq.aa offspring = p2q2 + 2pq3 + q4 = q2(p2 + 2pq + q2) =
q2(p + q)2 = q2(1)2 = q2.

Assumptions of the Hardy-Weinberg Principle
Mating is random
Allelic frequencies are the same in males and females
All the genotypes are equal in viability and fertility
Mutation does not occur
Migration into the population is absent
The population is sufficiently large that the frequencies of
alleles do not change from generation to generation by chance
Extension of Hardy-Weinberg to any number of alleles
Frequency of any homozygote
= square of allele frequency
Frequency of any heterozygote
= 2 x product of allele frequencies
15
Punnett square of random mating with three alleles
Multiple Alleles
Frequencies for multiple alleles can be calculated using the
Hardy-Weinberg equation by adding more variables.

For instance, in a situation involving three alleles (p + q + r =
1), the frequencies of the genotypes are given by:
(p + q + r)2 = p2 + q2 + r2 + 2pq + 2pr + 2qr = 1
17
Short tandem repeat (STR) loci contain between 2-9 bp repeats.
They typically contain alleles that are between 5 and 25 repeats.
The identity of an individual’s alleles can be determined by
performing PCR.
Primers must flank the region to be amplified.
The size of the product is proportional to the number of repeats.
A 5’ fluorescent tag can be added to one of the primers for
subsequent detection.
COmbined DNA Index System (CODIS)
Used by the FBI and paternity testing labs for human identity
testing.
13 STR loci plus the gender discrimination locus Amelogenin.
Multiplex PCR
Multiple PCR targets can be amplified in the same tube if their
products can be resolved.
Two ways to resolve amplicons:
Size: By placing the primers different distances away from the
STR it is possible to dictate the size range of the products.

Color: By adding a different colored fluorphore to the primer,
even products that overlap in size can be spectrally resolved.
20
Color Multiplex
By attaching different colored fluors to the primer, it is possible
to resolve overlapping sized amplicons.
We will be using 11 of the 16 loci from the Promega Powerplex
16
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CSI Fredonia Lab Writeup Guidelines
Introduction (40 points)
(consistent with) a suspect

based on matching with evidence.
ective pressure so that
Hardy Weinberg Equilibrium
applies.
probability of a specific
genotype in a population if allele frequencies are known.
cuss multi-channel fluorescence detection (Li-Cor DNA
analyzer)
Materials & Methods (25 points)
to cite lab manual.
lecular weight calculation
o Standard curve
o Unknown interpolation
the crime scene.

-matching suspects.
Data and Results (80 Points)
the sample in each lane.
Figure composition and formatting is important. See the
example figures at the end of
this rubric.
based on
the Ladder standards.
each suspect at the 11 loci
tested in the FergPlex11 reaction. This will be based on the
genotypes reported by your
classmates. You don’t have to show plots of every suspect from
the class.
pooled spreadsheet)
r each of your
samples using both the

Fredonia frequencies and the OmniPop frequency.
o Collect allele frequencies from the google spreadsheet
(Fredonia’s allele
frequencies)
o Use the FBI Caucasian population (column H, rows 72-110)
from the OmniPop
spreadsheet. Note that because the OmniPop data set does not
include Penta D,
do not use Penta D when performing the OmniPop analysis. Do
use Penta D for
the Fredonia genotype analysis.
o Report how the frequencies differ between the two data sets.
should realize that the
frequency that is being calculated is that of a genotype in a
population. It is based on
the allele frequencies that are observed. It is not how likely the
suspect matches
(because she matches 100%), but rather it is the probability that
we have implicated the
wrong person based on a chance match.

References (5 points)
Cite the lab handout and powerpoints, Butler 2006, and any
literature / websites that you
collect information from. Use the professional citation format of
your choice. I recommend the journal
Genetics, but others are also acceptable.

Figure 1: Multiplex Gel. Gel of FergPlex11 PCR reactions for
the 7 samples
analyzed by our group. IRD700 labeled PCR products are
displayed in green
and IRD800 products are plotted in Red. Detailed analysis of
each lane can
be found in figures 2-3.
S
a
m
p
le
1

S
a
m
p
le
2
N
o
D
N
A
La
d
d
e
r
La
d
d
e
r
S

a
m
p
le
3
S
a
m
p
le
4
S
a
m
p
le
5
S
a
m
p
le

6
S
a
m
p
le
7
Figure 2: Molecular Weight Standards. Electropherogram of
IRD700-labeled Molecular weight
standard bands between 100-460 bp. This ladder was used to
determine the genotype of samples 1-
4.

Figure 3: Suspect 12345. Electropherogram of FergPlex11
amplification from Suspect 12345 genomic
DNA. IRD700 labeled PCR products are plotted in green and
IRD800 products are plotted in Red.
Overlapping peaks are indicated in yellow.
460 bp 400 bp
364 bp
350 bp 300 bp 255 bp
204 bp
200 bp 145 bp 100 bp

Penta D
11/7
CSF1PO
13/12
FGA
24/23.2
D16S539
12/11
TPOX
11/8
D7S820
13/11
D8S1179
15/14
D13S317
15/15
vWA
16/15
D5S818
13/11
Amel
X/X

1
CSI: Fredonia – Determination of STR Genotypes by
Fluorescent Multiplex PCR.
Objectives:
1. Isolate genomic DNA from your own buccal cells using a
DNA swab
2. Set up a single PCR reaction with each DNA sample to
amplify 10 short
tandem repeat (STR) loci and one gender determination locus.
3. Understand the theory and practice of generating and
detecting fluorescent
PCR products.
4. Become familiar with the use of NIH ImageJ software for
image analysis.
5. Learn to build a molecular weight standard curve and perform
interpolation between points to
assess the molecular weight of unknowns.
6. Collect DNA from “suspects” in Jewett Hall and attempt to
match them to DNA collected at a crime
scene.
7. Report your confidence that you have identified the
perpetrator of the crime.

INTRODUCTION: The polymerase chain reaction (PCR) is a
method by which a small, defined
region of DNA can be synthesized from a minute amount of
DNA, as little as a single molecule, to
yield quantities of DNA sufficient for detailed analyses such as
gel electrophoresis or sequencing.
Today, you will collect your buccal cells using a DNA swab and
isolate your own genomic DNA
from these cells. You will use your DNA preparation to set up a
PCR reaction specific for 11
different loci. Your samples will be amplified, and the PCR
products will be analyzed by
polyacrylamide gel electrophoresis. The goal is to determine
the identity of a criminal from a
slate of suspects and assess the likelihood that you have the
correct perpetrator.
Fictional Premise of CSI Fredonia: A crime has been committed
in the new Science Center.
Over the winter break a trespasser broke into the science center
and walked across the freshly
poured floors in their dirty boots. As a result the floors in the
second floor hallway has been
ruined and must be replaced. University police arived on the
scene in time to see a single person

running from the worksite. While the perpetrator was not
apprehended, they did loose their hat
on the fence as they made their escape. Several hairs were
found in the hat with the follicles
intact. The police have provided us with DNA extracted from
these follicles in hopes that we can
identify the perpetrator and bring them to justice. It is up to you
to solve this hairy crime and
identify the person responsible for delaying the completion of
the Biology department's new
home.
The COmbined DNA Index System (CODIS) is a set of 13 loci,
each with multiple
tetranucleotide (4 bp) repeat alleles. This system is used by the
FBI to match evidence collected
at crime scenes with potential suspects. It also serves as a
mechanism to resolve paternity
disputes by comparing children with alleged fathers. It is
extremely robust and is capable of
generating genotypes that are able to identify individuals with
high probability. The figure to the

2
right shows the 13 loci and their location in the human
genome. An interactive version of this figure is available
at http://www.cstl.nist.gov/div831/strbase/fbicore.htm
These loci are analyzed by designing primers that flank
(are on either side of) the variable region. This allows the
PCR product to vary in size proportionally to the number
of repeats. The greater the number of repeats at that
locus, the larger the PCR product will be. This can be
ascertained by gel electrophoresis.
Multiplex PCR: The locus that will be amplified by PCR
is determined by the sequence of the primers that are
used. PCR primers have several important features that you
should be familiar with.
1. Two primers are necessary for PCR to occur. One is located
upstream of the region to be
amplified (also called an amplicon) and is called the forward
primer and one is
downstream and referred to as the reverse primer.

2. Primers bind to opposite strands of the template DNA. The
forward primer binds to the
bottom strand while the reverse primer binds to the top strand.
3. The primers are convergent. This means that the 3’ end of the
primers point towards
each other. Because DNA polymerase extends the 3’ end it is
necessary that it extend into
the amplicon and not away from it. During the extension phase
each primer must be able
to extend across the amplicon and synthesize the complement
for the other primer.
4. The primers are embedded in the final PCR product at the 5’
end of each strand.
Figure 2: The basis of STR allele discrimination when amplified
by PCR. Increased numbers of repeats result in a
longer PCR product. In this example at the vWA locus, the 10
repeat allele is 123 bp while the 13 repeat allele is
135 bp. It is important to appreciate that molecular alleles are
codominant. A heterozygote for the 10 and 13
alleles would produce two bands of 123 and 135 bp that could
both be detected. The discriminating feature is size.

Figure 1: The 13 CODIS Loci used for
forensic identity testing.
http://www.cstl.nist.gov/div831/strbase/fbicore.htm
3
Because the primers dictate the amplicon, it is possible to
design different size amplicons by
simply placing the primers closer or further apart from each
other. This allows researchers to
specify the range of sizes produced when amplifying an STR
locus (or any locus). It is also possible
to amplify multiple loci simultaneously in the same tube by
designing the primers such that the
resulting products do not overlap in size. We will use this
premise to amplify multiple different
loci in the same PCR reaction. Consider the set of 6 loci in
Figure 3. The primers for these 6 loci
have been designed to
generate PCR products

that do not overlap.
They range within the
region specified for
each locus, therefore if
a band appears in that
size range it can be
attributed to that locus
rather than one of the
other loci.
Further Multiplexing with Fluorescence: We have just learned
about multiplexing by size,
however it is possible to further discriminate between
amplicons of similar size by tagging each
with a fluorescent molecule. In PCR this is as simple as adding
a fluorescent tag to the 5’ end of
one of the primers (see figure 2). Modifications at the 5’ end do
not interfere with extension of
the 3’ end of the primer and therefore don’t affect their
efficiency in PCR. It does however allow

Figure 4: Fluorescent Multiplex amplicons. The amplicon in the
PowerPlex 16 kit from Promega amplify 16 loci,
grouped into three colors (the three rows). The loci bound by
the blue box are the CODIS loci, while the orange
box denotes two supplementary pentanucleotide STRs. The 11
loci to be studied in our lab are bound by the
green box. The IRD700 and IRD800 labeled loci are designated.
Figure modified from Promega corp.
Figure 3: Multiplex amplicons. The amplicon size for each of
six loci do not
overlap, thereby permitting simultaneous amplification and
allele discrimination.
Figure from www.nfstc.org
http://www.nfstc.org/
4

researchers to uniquely identify two products that are tagged
with fluorophores that emit
different wavelengths (colors) of light. Using this technology
“real forensic labs” can
simultaneously detect four (or more) different colored PCR
products that overlap in size. In our
lab we will be using two dyes called IRD700 and IRD800.
These dyes emit infrared light that
cannot be detected by the human eye, but can be resolved with
our electrophoresis system, the
Li-Cor Genetic Analyzer 4300. Figure 4 shows the 16 loci
contained in the PowerPlex 16 system by
Promega corporation. PowerPlex 16 contains all 13 CODIS loci
plus two pentanucleotide repeat
loci and the Amelogenin locus which can be used to distinguish
gender. We will be using a subset
of these loci that we’ll refer to (tongue-in-cheek) as FergPlex
11.
Allele Frequencies and Genotype Frequencies: All of the loci
that we are amplifying are
unlinked and thereby segregate independently. Only D5S818
and CXF1PO are located on the
same chromosome, however they are far enough apart that they
too segregate independently.

These loci are also not associated with any phenotypic
characteristics that could result in
selective pressure on any of their alleles. These conditions
allow us to make predictions about
genotype frequencies based on Hardy-Weinberg Equilibrium
(HWE) assumptions. Essentially
there are several frequencies that are important for this
exercise:
1. Allele frequency: This is the proportion of all alleles in the
population under consideration
that match the allele in question. In other words, the number of
a specific allele divided
by the total alleles in the population. As with all frequencies,
this number will be between
1 and 0. An allele that is fixed has a frequency of 1. An allele
that is lost has a frequency of
0. The sum of all allele frequencies for a given locus must equal
1.
2. Genotype frequency at one locus: Each individual has exactly
two allele at a given locus.
They can be the same (homozygous) or different
(heterozygous).
a. HWE predicts that a homozygous genotype will occur with a
frequency of p

2
where p is the allele frequency for the allele in question.
b. Heterozygous genotypes will arise in the population with a
frequency of 2pq
where p is the frequency of one allele and q is the frequency of
the other.
3. Genotype frequency at multiple loci: For loci that segregate
independently, the probability
of a genotype at one locus is an independent event from
genotypes at other loci.
Therefore to calculate an overall genotype frequency, you must
multiply the genotype
frequencies at each individual locus. This statistic is the
probability of a specific genotype
arising at random in a population with the allele frequencies
specified. If evidence is
matched with a suspect, this is the probability that the match is
due to chance and that
you have the incorrect suspect. By determining the genotype at
additional loci it is
possible to increase the confidence of establishing a correct
identity by reducing the
probability that the match has occurred by chance. These

frequencies are often reported
5
as odds (chance of 1 in 1,600,000 for example). Simply take the
reciprocal of the
genotype frequency to convert this statistic to odds ((6.25E-7)
-1
= 1,600,000).
Experimental Procedures:
A. Collection of buccal cells using a Catch-All DNA Swab:
Each group will isolate DNA from an
equal number of samples, including their own by following the
procedure described below.
Check each box as you complete the steps:
water before collecting her/his
buccal cells. Walk to the water fountain and rinse your mouth
twice. Lick the insides of
your cheeks to rinse off any bacteria. PLEASE DON’T SPIT IN
THE FOUNTAIN!!

Quick-Extract DNA solution from
the side bench.
the inside of your cheek.
Roll the swab about 20 times against the inside of each
cheek, making sure you move it
over your entire cheek. The more cells you collect, the higher
your yield of DNA will be.
-Extract DNA
extraction solution.
Rotating the brush between 5 and
10 times dislodges the cells from the brush.
swab while removing it from
the tube. This ensures that most of the liquid and cells remain in
the tube.
seconds. This ensures that
the cells and the solution are well mixed.
rs in
your e-mail address) or the
forensic ID for that sample using a black marker.

degrade the DNA or inhibit
the PCR reaction.
reactions.
genomic DNA suitable for
PCR amplification.
B. Setting up the FergPlex 11 multiplex reaction: Each group
will set up PCR reactions specific
for the 11 loci described above using the buccal cell genomic
DNA you just isolated.
prior to adding it to the reaction.
Set up an eppi (this is shorthand for eppendorf microcentrifuge
tube and will be used
6

henceforth) for each sample and label it with
nomic DNA.
Repeat this for each sample. Set the diluted
DNA on ice until it is called for.
will be genotyping. Do not separate the tubes. Keep them in
strips of 8 if possible.
your ID number. Make sure to mark the
tube on the neck rather than
on the top or conical portion. This will prevent the markings
from coming off.
all 22 primers necessary to
amplify the 11 loci in the FergPlex 11 reaction. Detailed primer
information is posted on
ANGEL. You will need to dilute the 5x concentrate to working
strength (1x) prior to adding
it to your PCR tubes.
the variable n in the following

formula.
mix. The 0.5 that is introduced into this formula is to
compensate for pipetting error
and thereby ensure that you will have adequate primer working
solution for each
sample.
each PCR tube. DO NOT
touch the bead with the pipette tip.
the matching ID and cap them.
BE SURE TO USE A NEW PIPETTE FOR EACH
INDIVIDUAL!
-2 sec).
Figure 5: Proper labeling of PCR tubes. Write
on the neck, NOT the top or conical portion.
Figure 6: The temperature profile used to amplify the PCR
products in the FergPlex 11 multiplex. Figure and

conditions modified from the PowerPlex 16 manual (Promega
Corp.)
7
C. Pouring a Denaturing Polyacrylamide Gel: The products of
our PCR reaction will be
separated on an extremely high resolution polyacrylamide gel.
This gel is different from the
agarose gels that you have run in the past in a few important
ways. First, it is made of a
polymer of acrylamide and N,N'-methylene-bisacrylamide
(colloquially “bis”). The monomeric
form of acrylamide is a neurotoxin. Therefore always wear
gloves when handling acrylamide
products. The polymerization of acrylamide and bis is initiated
by the addition of ammonium
persulfate and TEMED. These initiators generate free radicals
that convert acrylamide to a
free radical that reacts with other monomers to form a polymer.
The bis acrylamide is bi-

functional and forms crosslinks between adjacent acrylamide
chains. The second difference
between our gel and agarose
gels is that the denaturant
urea is incorporated into the
gel. The urea prevents the
two strands of a DNA duplex
from coming together. This
ensures that there is no
secondary structure in the
sample (a source of
heterogeneity) and facilitates
higher resolution. The gel is
prevent annealing. The final
difference is that this gel is
only 0.25 mm thick and is run
at over 1,000 volts.

the 6.5 % acrylamide solution from the fridge and
measure 20 mL in a graduated
cylinder. Pour this solution into a small beaker with a stir bar
and stir slowly. This will
allow the solution to warm to room temperature.
well to ensure that there are no
pieces of dry acrylamide or
dust. Clean the plates one last time with 70% ethanol and a
large kimwipe. Also clean a
comb and set it aside.
side of the plates should
face each other.
rig.
Figure 7: Acrylamide polymerization reaction. Acrylamide
monomers
are converted to free radicals by ammonium persulfate (APS)
and react
with other monomers or the end of an existing polymer. Bis
acrylamide
is bifunctional and acts as a crosslink between adjacent chains.
Figure
from http://sdspage123.blogspot.com/
http://sdspage123.blogspot.com/

8
of the casting
rig and then tighten the rails
well.
weighing out 100 mg of APS and
dissolving it in 1 mL of water in an eppi. This solution should
be made fresh right before
casting the gel.
acrylamide solution. Allow the solution
to mix for 15-20 seconds. You must work quickly now because
the polymerization
reaction has begun.
solution into the top of the
gel. Do not inject too quickly or air bubbles will form in the
gel. Prevent bubbles by
knocking on the front plate (like knocking on a door).

e gel area is full, lay the plates flat and insert
the flat side of the comb.
screws. This piece is easily broken
by over tightening. Only moderate pressure is necessary.
idify for about an hour.
D. Gel Prerun: Once solidified, the gel must be pre-run to allow
the gel to warm up, equilibrate
with the buffer, and to run out any residual initiators (APS and
TEMED).
laser has an
unobstructed view of the
gel through the glass plate. This is very important for the best
gel image.
place the lower reservoir
on the genetic analyzer. Don’t fill them yet.
10x) concentrate.
then pour the remainder of the

liter of buffer into the lower buffer reservoir.
from the top of the gel.
cover of the genetic analyzer.
ite
program and then prerun.
E. Sample Prep: The PCR samples must be denatured by mixing
with formamide and heating
them prior to loading on the gel. While the gel is prerunning,
prep your samples for loading
as follows:
of water into a clean eppi and
concentration of the PCR
products is too high and will result in overexposed and smeary
bands.
9
e diluted PCR reaction to an empty PCR

of stop solution. The stop solution is analogous to loading dye
that you have used in the
past with the exception that it contains the denaturant
formamide.
tubes.
immediately transfer the
samples to ice (called quenching). The fast quench prevents the
denatured strands from
having time to renature.
F. Gel Loading and Electrophoresis: Loading a 0.25 mm gel is
more difficult that you might
think. Follow these instructions to ensure a clean looking gel.
that no scraps of
acrylamide have become lodged between the plates at the top of
the gel. This is a major
source of frustration for beginners.
the top of the gel (about 2-3
mm below the top of the plate).

, therefore it is
necessary to periodically
rinse them out with a syringe filled with 1x TBE. Do this
several lanes ahead of where you
are loading.
directions in figure 8. This
prevents curvature at the edge of the gel (smiling) due to ion
imbalance.
every 4 lanes to permit accurate
Figure 8: Polyacrylamide Gel Loading. When loading, place the
sample into the void between the teeth of the
comb. The pipette tip will not fit into this space (it is only 0.25
mm wide), so it is necessary to allow the sample
to run down into the void. Because the buffer is warm, it will
cause the air in the tip above your sample to
expand and gently push the sample into the well. Don’t use the
pipette plunger to push the sample is as bubbles
will result causing sample mixing.
10
interpolation of unknown bands in our samples.

sample in case the gel needs to be
repeated.
will be posted to ANGEL for
subsequent analysis.
Gel analysis using Image J from the NIH
1. Download your gels from ANGEL. They are in the CSI:
Fredonia folder.
2. Open Image J from your flash drive.
3. Open the gel that you want analyze
4. Click on Image -> Color -> Make Composite. This ensures
that the image is in composite
mode which means that each color (red and green in our data) is
treated separately. You
will notice that turning the wheel on your mouse
or sliding the scroll bar at the bottom of the
image will change the channel at the top of the
window.
5. Click on the rectangle selection tool
6. Draw a rectangle around the first lane. It

should be somewhat narrower than the entire lane.
7. Click Analyze -> Gels -> Select first lane (or press Ctrl + 1).
This will designate the region
that you selected as the first lane in your gel.
8. Click on the lane selection (your cursor will look like an
arrow NOT a cross). Don’t select
anywhere else or you’ll have to start over! Drag this rectangle
over to the center of the
adjacent lane.
9. Click analyze -> Gels -> Select next lane (or press Ctrl + 2).
This will designate the second
lane.
10. Repeat step 9 for each of your lanes.
11. Ensure that the red channel is selected at the top of your
image (adjust with the scroll bar
at the bottom until it reads 1/3 (Red)
12. Click analyze -> Gels -> Gel Analyzer Options. Ensure that
the invert
peaks option is NOT selected.
13. Click Analyze -> Gels -> Plot Lanes and a density plot of
the red
channel from your gel will appear.

14. Click Edit -> Invert to change the lines to white and the
background
to black. This will be important for the overlaid image that we
will
work with shortly.
15. On the gel image move the slider to the green channel. It
should read 2/3 (Green).
11
16. Click Analyze -> Gels -> Re-plot Lanes It is important that
you click RE-plot. Plot lanes will
not work.
17. Invert the plot as described in step 13.
18. We will now overlay the two plot channels so they look like
they do on the gel. Click
Image -> Stacks -> Images to Stack. Then click Image -> Color
-> Stack to RGB.
19. Discard the black and white stack and save the red and
green plot image. This is an good
time to pause if you need to return to your
analysis later.

20. Click on the point selection tool.
21. Click on the first peak at the left in the plot
that corresponds to your standards.
22. Click Analyze -> Measure (or press Ctrl + M). This will
collect
information about the position of the point selector (and the
position of the peak) in a new window called “Results”.
23. Moving from left to right, collect the position of each of the
peaks by moving the point selector and pressing Ctrl + M at
each
peak (there should be 10 peaks).
24. Copy the data from the results window by selecting it with
the
mouse and copy it with Ctrl + C.
25. Open “CSI Data Analysis.xlsm” in Microsoft Excel. When
prompted be sure to allow
Macros to run.
26. Paste the data that you copied in step 24 into a blank area of
the
spreadsheet, then copy the X position data into the “Observed

Position” area under Molecular Weight Standards.
27. The spreadsheet will use these data to generate a standard
curve which will allow us to interpolate between the known
positions of the ladder to determine the molecular weight of an
unknown band.
28. Return to the plot in Image J and use the point selection tool
to
record the position of each of the red peaks in the lane that you
are analyzing. Make a note of any loci that only have a single
band and are therefore homozygous. This may be easier when
looking at the gel.
29. Copy the data from the results window into Excel. Enter the
position of the red bands into
the corresponding column labeled “Observed Position” under
the “Red Unknown Bands”
heading. If a locus was homozygous, then enter that value twice
at that locus.
30. Repeat steps 29-29 for the green unknown bands.
31. The spreadsheet will report the predicted molecular weight
of the unknown bands and
return the allele with the closest molecular weight.

12
Collaborative Analysis of Allele Frequencies
The genotypes that you have generated above will need to be
pooled across the class. We will be
using Google Drive to achieve this goal. The genotypes of each
individual will be entered into the
Google spreadsheet called “Allele Frequency Analysis”. To get
to your Google Drive, click on the
“Drive” link at the top of your Fredonia e-mail page (you must
be logged into your Fredonia e-
mail). Once there, click on the “Shared with me” link on the left
side of the Drive page. Under
that link, you should see the “Molecular Genetics 2013” folder.
The “Allele Frequency Analysis”
sheet should be visible therein.
1. Enter the sample ID at the top of the column.
2. Enter your name in the second row (analyst).
3. Copy and paste the genotypes determined on the “CSI
Data Analysis” spreadsheet into this spreadsheet. DO

NOT INCLUDE AMEL.
4. Once all of the data from the class has been entered we
can see the allele frequencies at all of the loci and then
calculate the probability of the perpetrator’s genotype
arising at random in the population.
hey, I need help with Lab report for my class ( Molecular
Genetic Lab) CSI report - I have 3 samples in a gel , I need you
to make ( Gel analysis using axel program ) then calculate the
probability of the perpetrators genotype arising at random in the
population and use the results and the pictures in the report ..
please cite the lab handout, powerpoint, the literature and every
swebsite you use to make it (I will send the handout, pp. &
lecture to u )it is due Feb 18. I want excellent work with no
PLAGIARIZE
***file name containing- CSI_Fredonia_2014_Handout.pdf
this is the handout > steps to follow for axle work , then use it
to write the report
****file name containing
CSI_Rubric_and_Example_Figures.pdf
this is the gaidline/example figure to do the report
****file name contaiunig CSI Fredonia 2014 Gel .tiff.pdf
( for axel work) I will send u 2 videos links that will help u
how to do analysis my 3 DNA sampels from the gel and the
other papers that he want us to use. when u done from step 31
from this file (CSI Fredonia Handout) please send me the
results and I will put it in a file called "Allele Frequency
Analysis " to share it with the other student results .. then i will

send it to u . then u can calculate the probability in the last step
in axle then u start doing the report..
the videos links;
http://www.youtube.com/watch?v=F2V6RcnLh80&feature=yout
u.be
http://www.youtube.com/watch?v=ws65HqNlJDA&feature=yout
u.be
I attached the Gel
and my samples just 3 from it ,
two of them are located after Ladder number 7 (last two)
the other one is located after Ladder number 8
name my samples in the report
A1 - AA
A2 - AA
H2 – AA
**** and this is axle sheet
CSI_Data_Analysis.xlsm
***also this is the last one for analysis
just follow the handout and u will find in the steps what to do
OmniPop200.1.xlsm
***CSI_Fredonia pwerpoint.pptx
cite every thing also the power point
s is the power point, it will help u specialty on this step ( to
calculate the the probability of the perpetrators phenotype
arising at random in the population ) use FBI info from file
"Allele Frequency Analysis Spreadsheet " if u can not use it just
send me the result and i will enter it on this sheet and send it
back to u to continue your work
**Allele Frequency Analysis 2014.pdf
"Allele Frequency Analysis Spreadsheet
***download it and it will work with you .
i shared this on Box.com

https://app.box.com/s/0durehgww80qothxd8j5
open the Gel folder from this tool by click on > file > open and
choose the Gel pic that I sent to u
if u need any thing just let me know

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