Review of DNA Extraction Methodologies and Guidelines for Protocol Development

Prepared by: Imogene Cancellare, University of Delaware, Panthera; Charlotte Hacker,

Duquesne University; Dr. Jan Janecka, Duquesne University; and Dr. Byron Weckworth,
Panthera

DNA extraction is required for many molecular biology applications, and many commercial kits
are available to isolate DNA from a variety of source materials. The quality and purity of the
extracted DNA should be suitable for the intended downstream analyses. These days, most labs
use commercial DNA extraction kits (rather than the traditional phenol-chloroform method),
which employ spin columns, for DNA isolation. Spin columns contain a silica resin that
selectively binds DNA, depending on the salt conditions and other factors influenced by
the extraction method. These kits are generally much easier and faster to use than traditional
methods, and do not require significant expertise. However, regardless of the kit used, and/or if
the lab must optimize a protocol based on available supplies and reagents, it is important to
understand the basic criteria that any method of DNA isolation must meet to determine whether a
protocol is a suitable alternative, particularly for fecal DNA because of the low quantity and
quality of the DNA.
For any sample type, a kit or protocol should satisfy the following criteria: 1. efficient
extraction of DNA, 2. successful removal of contaminants, 3. production of sufficient amount of
DNA for downstream workflow, and 4. isolation of high quality and high purity DNA. The
following is a review of each basic step involved in all DNA extraction methods, and guidelines
for selecting protocols and reagents to ensure the above criteria are met.

DNA extraction methods comprise three main steps:

1. Lysis of cell walls and membranes to release DNA into solution
The first step of DNA isolation is the disruption and lysis of cell walls and plasma membranes of
cells and organelles. Lysis buffers contain a high concentration of chaotropic salts. Chaotropes
have two important roles in nucleic acid extraction. First, they destabilize hydrogen bonds, van
der Waals forces and hydrophobic interactions, leading to destabilization of proteins, including
nucleases. Second, they disrupt the association of nucleic acids with water (which provides
optimal conditions for transfer to silica if spin columns are being used). Incomplete lysis
significantly reduces DNA yield. In addition to chaotropes, a detergent is often present in the
lysis buffer to aid protein solubilization and cell lysis. This step also involves a protease for the
digestion of cellular proteins that contaminate target DNA. Proteinase K works best under
protein denaturing conditions (i.e. in denaturing lysis buffer). There are many lysis and protease
solutions available:
• Buffers ATL and AL (from Qiagen kit): cell lysis solutions that break open tissue, cell, and
nuclear membranes. Contains chaotropic agent guandinium chloride.
• Homemade lysis buffers commonly contain the following: 0.5% SDS, 8% PVP-10 (opt, 250
mM NaCl, 25 mM Na-DETA, 200 mM Tris-HCl; pH 7.5
• Proteinase K, 20ug/mL: a broad-spectrum serine protease. Very efficient in digesting proteins
during nucleic acid preparation. Works by catalyzing the breakdown of cellular proteins by
splitting them into smaller peptides and amino acids.

Description of kit-specific additions (not necessary for successful extraction if creating a

homebrewed protocol):
• InhibitEX solution (Qiagen) is designed to remove inhibitors from samples which can impact
down-stream processing, and may be added prior to Proteinase K. The exact composition of
this solution is proprietary information.
• Some commercial kits that do not use columns utilize a high-salt buffer (e.g., Protein
Precipitation Solution, Qiagen) during the lysis step to lower the solubility of proteins and
allow protein precipitation so they can be pelleted during centrifugation.
• Some commercial kits use RNase A solution, which ensures that cellular RNA is digested
during lysis.
• Phosphate-buffered saline (PBS) (Gentra Puregene) is used to isolate white blood cells from
blood or other cells from fluids and prevents rupture of cells, balancing the salt concentration
around surrounding cells. Contains 50 mM potassium phosphate, 150 mM NaCl; pH 7.2.

2. Purification of DNA by precipitating proteins and polysaccharides

Following cell lysis, proteins in the sample are precipitated out of the solution and removed
following separation by centrifugation. Alcohol precipitation is used for concentrating, desalting,
and recovering nucleic acids. Precipitation is facilitated by high concentrations of salt and the
addition of either isopropanol or ethanol. DNA is less soluble in isopropanol and allows
precipitation of lower concentrations of nucleic acids than ethanol, especially if incubated at low
temperature for longer periods of time. This can be performed at room temperature, which
minimizes co-precipitation of salt that interferes with downstream applications. However,
because isopropanol involves the co-precipitation of salts, a second wash with ethanol is
necessary. Ethanol is particularly efficient at precipitating polymeric nucleic acids and removing
chaotropic salts, and ideal for precipitating very small DNA fragments. Additionally, for
protocols using spin columns, the addition of ethanol further enhances the binding of nucleic
acids to the silica in the spin column. Different extraction methods may use ethanol or
isopropanol (or both) in washing buffers.
The percentage and volume of ethanol used are critical for optimal extraction. Too much
ethanol will precipitate degraded material and small non-specific organisms, which will
influence UV260 readings during DNA quantification. In contrast, too little ethanol may impede
removing salts from the precipitate, which can affect down-stream reactions. It is therefore
important to follow the recommendations of the kit or protocol being used, and to use fresh
ethanol. If you suspect that degraded DNA is inflating your absorbance readings during
quantification, consider re-optimizing ethanol concentration.
DNA purification generally involves two wash steps to remove residual protein and salt.
The first wash is either isopropanol, or an ethanol-based solution that includes a low
concentration of chaotropic salts to remove residual proteins and pigments (many names, i.e.
Wash buffer 1). This is always followed with an ethanol wash to remove co-precipitated salts
(e.g. Wash buffer 2), which is crucial for increasing DNA yield and purity (residual salt
contributes to high A230 readings and thus low 260/230 ratios). Residual contaminants,
including salts, may also interfere with accurate quantification, and may result in reduced
260/280 ratios. Some kits may recommend two ethanol washes.
There are several versions of ethanol or isopropanol-based washing buffers to aid purification:
• Buffer AW1 and AW2/ Wash Buffer 1, 2 (Qiagen): Wash solutions that remove contaminants
from the DNA attached in the column membrane.
• Homemade washing buffer I (first washing) for protocols without isopropanol should contain
the following:10 mM NaCl, 10 mM Tris-HCl pH 6.5, 80% ethanol.
• For kits and protocols using isopropanol, 100% isopropanol is used for the first washing.
• Homemade washing buffer II (second washing) comprises only 70% to 95% ethanol.
Description of kit-specific additions (not necessary for successful extraction if creating a
protocol):
• Glycogen solution 20mg/mL (Gentra Puregene Kit): During isopropanol precipitation,
Glycogen Solution acts as a nucleic acid carrier and helps to efficiently precipitate small
amounts of DNA. In addition, it facilitates visualization of the DNA pellet. The exact
composition of this solution is proprietary information. If building a protocol, note that
Glycogen Solution does not work in combination with Qiagen’s silica membrane column-based
technologies, e.g., RNeasy, DNeasy, QIAamp, etc.
• Some column-based kits may use a binding buffer to bind DNA to filter paper discs.
Homemade binding buffer contains the following concentrations: 2M guanidine
hydrochloride, 75% ethanol.
3. Precipitation of DNA and resuspension in a buffer
Most protocols include a drying step to remove residual ethanol, either by drying the spin
column (for column-based methods) or by evaporating ethanol from the sample tubes. This step
is essential for a clean eluent because the presence of ethanol will prevent full hydration, elution
and interfere with downstream protocols.
DNA is resuspended from spin columns or dried sample tubes with the addition of an
elution buffer. 10 mM Tris buffer will hydrate the DNA and result in higher quality elution than
nuclease-free water, which has a lower pH and may not be adequate for hydrating high molecular
weight DNA. For maximal DNA elution, it is recommended to allow buffer to stand in the silica
membrane before centrifugation (column-based protocols), or incubation to dissolve DNA. For
applications requiring intact high molecular weight DNA, elution buffer is preferred over water-
based resuspension.
There are several elution buffers:
• Buffer AE and ATE (Qiagen): A solution that elutes the DNA from the membrane and allows
stable storage of DNA for years in the refrigerator or freezer.
• DNA Hydration Solution (Gentra Puregene): Same purpose as above. 10 mM Tris, 1 mM
EDTA; pH 7–8.
• Homemade elution buffers should contain the following concentrations: 10 mM Tris-Cl, 0.5
mM EDTA (optional); pH 9.0.

Considerations for all reagents

Regardless of the kit or protocol used, it is important to prepare and store reagents according to
supplier recommendations. Many buffers can be stored at room temperature, but certain steps
require cooling on ice. It is also important to use the amounts of sample material indicated in the
protocol, as limitations may vary based on the kit and reagents used.
For column-based extractions, avoid touching the silica membrane with the pipet tip.
Extra centrifugation time may be necessary for scat samples containing debris to ensure liquid
flows through the column (columns may change color, but eluate should be clear).
For some downstream applications, concentrated DNA may be required. Elution with
volumes of less than 200 µl increases the final DNA concentration in the eluate significantly, but
this slightly reduces overall DNA yield. For column-based methods, following the first elution of
200 µl, a second elution in a new sample tube of 100 µl can increase overall DNA yield.
Additionally, warming the elution buffer to promote evaporation (including by speedvac) can
increase yields by further concentrating the DNA.
Aggressive vortexing can shear target DNA, as can rapid hand mixing (e.g., DNA
precipitation with isopropanol and ethanol), so gentle inversion and pulse-vortexing is
recommended after lysis steps to minimize mechanical degradation of target DNA.
It is the responsibility of the lab to ensure that the reagents and storage buffers used are
appropriate for the desired sample processing method.

Building an ad hoc protocol

Silica column-based commercial extraction kits are a good choice for fecal DNA extraction
because each step in these protocols are optimized to account for the low quality and quantity of
fecal DNA, and troubleshooting is relatively easy when improved yields are required. When
commercial kits are not available, however, the user must decide if a protocol can be built to
mimic a spin column method, or if isopropanol/ethanol methods using hand mixing are more
appropriate. If silica membrane spin columns are available, and the user can mix appropriate
substitutes for a given commercial kit, the Qiagen scat extraction protocol is recommended.
However, if a comparable commercial kit is unavailable, manual processing using modified
salting-out precipitation methods are an ideal substitute due to the simplicity of protocol
optimization and availability of reagents or corresponding homemade substitutes. These methods
generally include isopropanol and ethanol-based washing buffers during the washing steps
(reagents described in this document are used in both extraction methodologies), and do not
require columns. Below is an example of a manual salting-out precipitation method that can be
modified for ad hoc protocols.

Untested potential protocols for scat

For labs unable to get Qiagen stool kits, a Gentra kit or the salting out method on scat may be a
viable alternative. The authors have not tested this directly, so it is recommended to test these
methods on samples first before using them. For salting out method protocols, see Miller, Dykes,
and Polesky 1987 (provided below) as well as https://openwetware.org/wiki/DNA_extraction_-
_Salting_Out .

Purification of archive-quality DNA from fecal cells in suspension using the Gentra®
Puregene® Tissue Kit
Equipment and reagents
Phosphate-buffered saline (PBS) = 50 mM potassium phosphate, 150 mM NaCl; pH 7.2
Cell lysis solution (0.5% SDS, 8% PVP-10, 250 mM NaCl, 25 mM Na-DETA, 200 mM Tris-
HCl pH7.5)
Elution buffer-10 mM Tris-Cl, 0.5 mM EDTA; pH 9.0.
Protein precipitation solution
100% isopropanol
70% ethanol (do not use denatured alcohol)
Pipets and pipet tips
1.5 mL microcentrifuge tubes
Microentrifuge
Water bath or heat block
Crushed ice
Absorbent paper/kimwipes™
Recommended: glycogen solution 20mg/mL
Optional: RNase A solution

Procedure:
1. Dilute 1–2 g stool in 10 ml PBS, and let the debris settle.
2. After the debris has settled, transfer 100 μl of the supernatant to a clean 1.5 ml
microcentrifuge tube.
3. Add 500 μl Cell Lysis Solution to 100 μl fecal cells in PBS, and mix by pipetting up and
down.
Note: If the sample has a high protein content, 550 μl Cell Lysis Solution may be added
to 50 μl sample.
4. Add 3 μl Proteinase K (20 mg/ml) and incubate lysate at 55°C for 1 h to overnight.
If you wish to include an optional RNase treatment, go to step 4a, otherwise proceed
with step 5.
4a. Add 3 μl RNase A Solution to the cell lysate, and mix by inverting the tube 25 times.
5. Quickly cool the sample to room temperature (15–25°C) by placing on ice for 1 min.
6. Add 200 μl Protein Precipitation Solution to the cell lysate, and vortex vigorously for 20s
at high speed.
7. Incubate on ice for 5 min.
8. Centrifuge at 13,000–16,000 x g for 3 min. The precipitated proteins should form a tight
pellet. Repeat if pellet not well-formed.
9. Pipet 600 μl isopropanol into a clean 1.5 ml microcentrifuge tube.
10. Recommended: Add 1 μl Glycogen Solution (20 mg/ml).
11. Add the supernatant from step 9 by pouring carefully. Make sure not to dislodge the
protein pellet when transferring the supernatant.
12. Mix by inverting gently 50 times and incubate at room temperature for 5 min.
13. Centrifuge at 13,000–16,000 x g for 5 min. The DNA may be visible as a small white
pellet, depending on yield.
14. Carefully discard the supernatant. Drain the tube on a clean piece of absorbent paper,
taking care that the pellet remains in the tube.
15. Add 600 μl of 70% ethanol, and invert gently several times to wash the DNA pellet.
16. Centrifuge at 13,000–16,000 x g for 1 min.
17. Carefully discard the supernatant. Drain the tube on a clean piece of absorbent paper,
taking care that the pellet remains in the tube. The pellet might be loose and easily
dislodged.
18. Allow DNA to air dry at room temperature for 10–15 min. Make sure tube is dry before
proceeding.
19. Add 20 μl DNA Hydration Solution. 100-200 μl of elution buffer is recommended to
ensure enough sample is available for downstream analyses. Low yields can be increased
by concentrating the sample.
20. Incubate at 65°C for 1 h to dissolve the DNA.
21. Incubate at room temperature overnight with gentle shaking. Ensure tube cap is tightly
closed to avoid leakage. Samples can then be centrifuged briefly and transferred to a storage
tube.
 Scat Collection for Host/Prey DNA Protocol
Prepared by: Charlotte Hacker, Duquesne University; Imogene Cancellare, University of Delaware,
Panthera; Dr. Byron Weckworth , Panthera; and Dr. Jan Janecka, Duquesne University

Sample Tube Preparation:

● Scat can be collected and stored in the following options:
o 15mL plastic tube with 7mL of color indicating drierite/silica desiccant (standard
species, individual identification, and diet projects or studies where space is
limited).
o 50mL plastic tube or specimen collection cup (or larger) half filled with
drierite/silica (23mL or equivalent) (standard species, individual identification, and
diet analysis projects where additional extractions needed).
▪ Notes: Various preservatives can be used to store scat samples -
● While effective, ethanol is discouraged as it can leak out of the
tubes and wash off identifying markings on the tube, and creates
difficulties when shipping samples.
● DET buffer must be prepared freshly and accurately, which may
not be logistically feasible in remote field conditions.
▪ A Kimwipe/tissue is recommended to separate desiccant from the scat
sample in smaller sized tubes to avoid the silica/drierite from getting on
the scat and/or contaminating it.
Scat Collection:
Sample Labeling –
● Label tube or cups with permanent marker using the following nomenclature [YEAR-
MONTH-DAY-COLLECTOR’S INITIALS-SCAT NUMBER].
● Example:
o First scat collected on June 1, 2020 by Charlotte
Hacker: 2020-06-01-CH-01
o Second scat collected on June 1, 2020 by Charlotte
Hacker: 2020-06-01-CH-02
▪ The numeric order of scats starts over each
new day of collection.
▪ Each collector uses their own numeric scale
starting from 01.
● Use the unique sample label on the cup to label a waypoint
Labeling for sampling collection.
for the scat location in the GPS.
● This system ensures that each scat has its own unique
field name and provides basic information for reference later on if needed, such as date
and collector.
Filling Out Sample Sheet –
● Fill out the corresponding sample
sheet with the information
pertinent to the sample being
currently collected.
● Set an empty labeled tube near
scat, but not touching it, with the
label facing upwards, and take a
picture prior to collection. Recording information prior to Picture of scat sample with tube
sample collection. and name adjacent.

Filling out sampling sheet example:

Date Sample Tran- Species Diameter Age Sign, Feature Nearest GPS/ Notes
ID sect Age, Landmark Elevation
Proximity

2020/06/01 2020- 01 Snow ~3cm old Fresh Ridge- Boulder 35.513 By

06-01- leopard scrap line ~3m 98.347 yak
CH-01 1m high 4442m skull
away
2020/06/01 2020- 01 Snow ~5cm fresh Old Rocky Set of 35.512 Grass
06-01- leopard pugmark Outcrop prayer 98.252 in
CH-02 2m flags 4472m scat
away
Definitions of each sampling sheet heading:
Heading Definition
Date Date the sample was collected.
Sample ID The field name of the sample as written on the tube.
Transect The transect number of which samples are being collected on.
Species The species the collector believes the scat to originate from.
Diameter The size in cm of the scat across its width as judged by eyesight.
Scat Age The relative age of the scat.
Fresh – high or some moisture content, dark in color, may or may not have odor.

Old – no moisture, discolored (faded or bright white), outside casing may be missing.
Sign, Age, Signs of carnivore presence near the sample, how old the signs appear, and their
Proximity distance to the sample. Examples include scrape, urine, latrines, claw rake,
pugmark, and scent spray.

Scrape – Results from snow leopards digging their

hind legs down and back into the ground, displacing
soil and loose earth.

Pugmark – A distinct footprint in the surface of the

ground. Can be identified down to species if clear
enough.

Latrine – A site used by multiple snow leopards or

other carnivores. Beneficial for detecting multiple
individuals living in an area at one site.

Feature A geographic feature for the landscape where the scat was found. Examples include
ridgelines, saddles, river drainage bottoms, flat grasslands, and rocky outcrops.

Ridgeline River Drainage Bottom Flat Grassland Rocky Outcrop

GPS and The GPS coordinates of where the scat was collected in decimal degrees and its
Elevation elevation in meters.
Notes Any additional information that may be useful.
Collection –
● It is imperative that bare hands do not touch the scat. This could lead to contamination
of the next scat sample with the previous one. A new tool for collection should be used
for every scat sample. Latex gloves are ideal, but rocks and sticks can also be used if
gloves are unavailable.
● Using sterile tweezers, fresh gloves, or rocks and sticks, break off/cut a full-cross-section
of the scat. Chunks of scat (full circumference and diameter) that appear freshest/less
dry are best as the DNA is less likely to be degraded and are more useful for diet
analysis. Place the scat into the tube.

Scat being broken apart and Scat being broken apart and Scat being broken apart and
collected with gloves. collected with a rock. collected with tweezers.

● Do not stuff the scat tightly into the tube. Make sure
there remains airspace between the scat and the lid of
the tube. This is so the desiccant can properly dry out
the scat to preserve the DNA inside the scat.
● Remove gloves or set down tools used to handle the
scat.
o Dispose of gloves properly by putting them in a
receptacle bag and then putting that bag in the
appropriate bin after the sampling session
Sample put into tube with airspace
(label and store separately from clean gloves). to ensure it dries properly.
● Place the cap back on the tube securely.
● If using tweezers, sterilize between samples with fire by using a lighter to burn off any
scat or hair on the tweezer for at least 5 seconds (let
cool before packing up tools). Do not touch the end of
the tweezers (the part used to collect the scat) at any
time with your bare hands.
● Place the tube in the collection bag.
● Avoid collecting the entire scat if not necessary in order
to maintain the chemical and visual cues scat provides
within a habitat. Sample marked with nail polish to
avoid recollection in future
● If sampling the same transect repeatedly, mark the sampling efforts.
remainder of the scat with nail polish so that it is not
collected during the next sampling session.
Data Entry –
● Entering data from field sheets into an electronic format relatively soon after sampling
sessions is helpful for organizing samples and preparing them for transport.
● At the end of every field day, take a photo of the datasheet for temporary backup in
case the datasheet is lost or destroyed. This covers the lag time between field collection
and entering the data electronically.
● Open up an excel spreadsheet and enter in the information on your sampling sheets as
written. Save file.
o Note: the names labeled on the collection cups in the field are helpful for
discerning date and collector, but are not practical for lab work later on when
sample names must be written repeatedly on the small surfaces of
microcentrifuge tubes. It is helpful to add an associated lab name to the
collection tubes with the samples.
▪ Line up your cups on a flat surface in order of date, collector, and then
collection number.
▪ Relabel each tube in order with a short prefix related to the sample site
followed by a number. Write this name on both the side of the tube and
its cap. Continue onward for the entire sampling session, picking up each
numeric value from where you left off prior.
▪ Add a “Lab Name” column into your Excel spreadsheet and enter in the
name of each sample corresponding with its field name.
▪ Example:
Field Name Lab Name
2020-06-01-CH-01 DUL01
2020-06-01-CH-02 DUL02
2020-06-01-IC-01 DUL03
2020-06-01-IC-02 DUL04
2020-06-01-IC-03 DUL05
2020-06-02-CH-01 DUL06
2020-06-02-CH-02 DUL07
2020-06-02-CH-03 DUL08

Sample Transport:
● Collected samples should be kept in a cool, dry place after
collection until they can be transported.
● Transport of samples should adhere to local government
and country regulations.
● Place sample tubes in a sealed bag. Label the outside of the
bag with name, material, and number of samples in bag.
● If multiple bags accumulate, place them in a larger box or
sturdy bag. Samples organized, labeled, and
packed for transport.
● All boxes should be reinforced with adequate tape to
ensure samples are secured and arrive safely.
Sample Storage:
● Upon return to the laboratory space, proper sample
storage ensures DNA degradation will not occur.
● Room temperature storage is possible so long as the
ambient temperature is stable.
o Labs that do not have reliable temperature control,
or those which are kept above room temperature
(~20°C), are not adequate for room temperature Samples stored in organized rows
storage at stable room temperature.
o Samples as old as 10 years stored at stable room
temperatures have resulted in successful DNA
extraction when stored in this manner
● Refrigerator or freezer storage should be considered if the
laboratory does not have consistent temperature control.
o Freezing samples halts microbial activity that could
further degrade DNA.

Samples stored in laboratory freezer.

Samples stored in urine specimen cups

with lab and field ID.
 Scat Collection for Host/Prey DNA
Pre-Collection Add silica desiccant Gather and organize
beads and tissue to collection materials.
collection container.
Date Sample
15mL
Transect Age Features GPS Notes
2019- 2019-
06- 06- old Out- 35.727 By yak
01 01- 06 crop 96.874 carcass

CEH01

10mL

5mL

L
15m

L
10m

Silica beads
5mL should take up Materials include collection
just under half of container, gloves, data
total container sheet, GPS unit, and
volume. permanent markers.

Label collection Fill out data collection Take picture of empty

container and lid using sheet. labeled collection
Collection

permanent marker. container next to scat.

Date Sample Transect Age Features GPS/Elevation Notes

2019- 95.24891/
2019/ 06-01- 3 1m from
old ridgeline 33.8735 yak carcass
06/01
CEH01

2019- 95.2133/
2019/ 06-01- 3 flat small creek

2019-06-01-CEH01 06/01 fresh grass 33.8930 10m away

CEH02

2019- rocky

2019-06-01-CEH01
2019/ 95.2265/ pug mark
10mL

06-01- 3
15mL

old outcrop 15mL

5mL

06/01 33.8065 1m away

CEH03

10mL

5mL

The following format should be

used in labeling–
[YEAR-MONTH-DAY-COLLECTOR’S INITIALS-SCAT NUMBER]

Dispose of gloves and Place piece of scat Put on gloves and

put container with scat into collection break off a full cross-
into collection bag. container, remove section of the scat
gloves, and secure lid. sample.
2019-06-01-CEH01

15mL

Chunks of scat that are

10mL
Do not stuff container full of fresher/less dry are best as the
scat. Scat must be loose so DNA is less degraded. Can use
-0
20
19
5mL
desiccant can dry out the scat sticks/rocks to handle scat if no
gloves available. Do not touch
6

in order preserve the DNA.

-0
1-
C
E
H0
1

scat with bare hands.

Keep labeled, organized Store samples long-

Post-Collection

samples in a cool, dry term at a consistent

place until sent or room temperature,
transported to lab. fridge, or freezer.
CEH01
06-01-
2019-

CEH01
06-01-
2019-

CEH01
06-01-
2019-
CEH01
06-01-
2019-

CEH01
06-01-
2019-

Be sure to
adhere to all
CEH01
06- 01-
2019-

CEH01
06- 01-
2019-

CEH01
06- 01-
2019-

CEH01
06-01-
2019-

CEH01
06-01-
2019-

0-
ples 202
Scat sam
shipping and
to
CEH01
06-01- EH07
6-01-C
2020-0
CEH01
06- 01-
2019-

CEH01
06- 01-
2019-

CEH01
06-01-
2019-

CEH01
06-01-
2019-

transportation
regulations.
CEH01
06-01-
2019-

CEH01
06-01-
2019-

CEH01
06-01-
2019-

CEH01
06- 01-
2019-

CEH01
06- 01-
2019-

Created By: Charlotte Hacker, Duquesne University; Imogene Cancellare, University of Delaware, Panthera; Dr. Byron Weckworth, Panthera; Dr. Jan E. Janecka, Duquesne University