**USGS/BRD**

**ALASKA SCIENCE CENTER**

**MOLECULAR ECOLOGY LABORATORY**

**Lab 102 and 103, 1011 E. Tudor Road**

**Anchorage, AK 99503**

#### (907) 786-3582; Fax (907) 786-3636

####### Sandra Talbot, Research Geneticist (Wildlife) Judy Gust, Geneticist

office: (907) 786-3683 office: (907) 786-3949

sandy_talbot@usgs.gov judy_gust@usgs.gov

Kevin Sage, Geneticist John Pearce, Wildlife Biologist

office: (907) 786-3975 office: (907) 786-3893

kevin_sage@usgs.gov john_pearce@usgs.gov

Talia Wiacek, Biological Science Technician

office: (907) 786-3494

talia_wiacek@usgs.gov

######## DNA SAMPLING PROTOCOLS

# Sample Collection Supplies

1\. Select the appropriate sample collection box based on the type of
sample to be collected.

+----------------------+-----------------------+----------------------+
| **Sample Type**      | ##### Collection Box  | #####                |
|                      |                       | # Short-term Storage |
+----------------------+-----------------------+----------------------+
| Blood                | Blood Buffer          | Room Temperature     |
+----------------------+-----------------------+----------------------+
| Blood                | Filter Paper in       | Dry, Room            |
|                      | Envelopes             | Temperature          |
+----------------------+-----------------------+----------------------+
| Blood quills         | Tissue Preservation   | Room Temperature     |
|                      | Buffer                |                      |
+----------------------+-----------------------+----------------------+
| Bone                 | Envelopes or Dry      | Dry, Room            |
|                      | Microtubes            | Temperature          |
+----------------------+-----------------------+----------------------+
| Egg shell membranes  | Envelopes or Dry      | Dry, Room            |
|                      | Microtubes            | Temperature          |
+----------------------+-----------------------+----------------------+
| Feathers from museum | Envelopes             | Dry, Room            |
| skins                |                       | Temperature          |
+----------------------+-----------------------+----------------------+
| Feathers from nests  | Tissue Preservation   | Room Temperature     |
|                      | Buffer                |                      |
+----------------------+-----------------------+----------------------+
| Feathers from nests  | Envelopes             | Dry, Room            |
|                      |                       | Temperature          |
+----------------------+-----------------------+----------------------+
| Fin Clips            | Ethanol               | Room Temperature     |
+----------------------+-----------------------+----------------------+
| Fin Clips            | Filter Paper,         | Dry, Room            |
|                      | Envelopes or Dry      | Temperature          |
|                      | Microtubes            |                      |
+----------------------+-----------------------+----------------------+
| Hair                 | Envelopes or Dry      | Dry, Room            |
|                      | Microtubes            | Temperature          |
+----------------------+-----------------------+----------------------+
| Muscle               | Tissue Preservation   | Room Temperature     |
|                      | Buffer                |                      |
+----------------------+-----------------------+----------------------+
| Scat                 | Ethanol               | Room Temperature     |
+----------------------+-----------------------+----------------------+
| Scat                 | Silica Beads/Gel      | Room Temperature     |
+----------------------+-----------------------+----------------------+
| Teeth                | Envelopes or Dry      | Dry, Room            |
|                      | Microtubes            | Temperature          |
+----------------------+-----------------------+----------------------+

2\. Check to ensure that your sample collection kit contains numbered
microtubes (i.e., 1 to 100), a Sample Collection Form, and a Sharpie
marker. Additionally, some kits may contain sampling scissors and
forceps.

3\. On the Sample Collection Form, record data associated with the
respective sample (i.e., species, sample ID or band number, collection
location, collection date, sex and age if known, and collector).
Additional data may be required, depending upon study.

4\. Label the outside of the box (i.e., species, collection location,
collection dates, collector name, and contact telephone number).

**[Dropping Off Samples]{.underline}**

1\. Return samples to the Sample Drop Box located in rm. 102 (Molecular
Ecology Lab) of the Alaska Science Center, 1011 E. Tudor Rd., Anchorage,
AK 99503. Samples may also be returned by regular mail.

2\. Record samples in the *Samples Received Log*.

3\. Complete *Sample Data Entry Form* for entry into the BIOTA Inventory
system.

## Collection of Materials for DNA Analysis

# Blood *(Blood/1 Vial/Blood Buffer/Room Temperature)*

For birds, blood is the preferred tissue type for nuclear DNA work, but
less valuable than muscle for work that includes mtDNA analyses. We have
provided 1.5 mL eppendorf tubes with a blood buffer storage solution
(Longmire Buffer). The chemicals are not toxic, but this solution should
not be ingested. The buffer and any blood/buffer combination can be
stored at room temperature until you return from the field, at which
point they should be frozen. Only a few drops of blood are needed from
each bird, enough to turn the buffer red (usually about 5 drops). Blood
can be obtained by pricking the tarsus vein and transferring the drops
to the tubes by way of capillary tubes **(DO NOT USE heparinized
tubes)**. Using a capillary bulb, blood can be blown from the capillary
tube into the buffer after collection. If the blood clots inside the
capillary tube, break off the tube inside the buffer vial and just leave
it there. Blood can also be collected from the brachial or jugular vein
with a sterile syringe. Use a new, sterile syringe for each individual.

**[Blood on filter paper]{.underline}** *(Blood/Whatman Filter
Paper/Dry/Room Temperature)*

5-6 drops of blood can be placed on filter paper (Whatman). The paper
should be kept separate from other samples to avoid contamination. Allow
the damp filter paper to dry and store separately in either: (1) a
ziploc bag with silica gel, or (2) a separate envelope.

**[Blood Quills]{.underline}.** *(Blood Quills/1 Vial/Tissue
Preservation Buffer/Room Temperature)*

Blood quills are put into tubes containing tissue preservation buffer
provided by our lab. Pull two or more blood quills from the bird. We
recommend sampling wing coverts rather than emerging primaries and
secondaries, which are more critical to flight. We extract DNA from the
bloody "skin end" of the quill, so if the quills will not fit into the
tube, trim off the feather tips, leaving the bloody ends (calamus) to
put into the tube.

We strongly suggest that latex gloves be used when sampling, and that
instruments be cleaned with 10% bleach between sampling. This prevents
between-sample contamination and protects the collector from infectious
diseases and any preservatives that may have been used in the skin's
preparation.

Tubes can be stored at ambient temperature for shipping.

# Egg Shell Membranes *(Egg shell membranes/1 Envelope/Dry/Room Temperature)*

DNA yields from eggshell membranes are very good, provided there is
vascularization on the membrane. The easiest field technique is to
collect each membrane and place it in a separate plastic bag: placing
all membranes in the same bag causes cross contamination of samples. We
do not use the hard shell at all, so that portion can be left in the
field. Do not store feathers and eggshells from the same nest in the
same bag. Give the nest a number, and then label each feather or egg
sample with that number (e.g., nest number 100 has feather sample number
100 and membrane sample numbers 100(1), 100(2), etc.).

**[Feathers from nests]{.underline}.** *(Feathers/1 Envelope/Dry/Room
temperature)*

We get the best results with contour or tail wing feathers (those with a
substantial sheath or rachis) deposited in nests or shed by birds during
molt. The DNA is actually in the calamus, so feathers without the
calamus cannot be used to extract DNA. We are unable to obtain DNA from
down feathers. Please collect as many contour feathers from each nest as
possible (we use 5 feathers per DNA extraction from geese and at least
that many for passerines, but like to have extra in case it does not
work the first time around). Feathers can be removed after the nest has
failed or hatched, or when first discovered if you don't plan to revisit
the nest. Keep feathers dry after collection, since moisture can cause
decay of feathers and subsequently the DNA. Place feathers in paper
envelopes or, if bone dry, in plastic bags. **Store feathers from
different nests in separate bags/envelopes**. Feathers do not need to be
frozen. Envelopes can be placed in a plastic bag with 2 Tbsp. silica gel
to aid in maintaining a dry environment. {See Pearce et al. (1997) for
additional considerations when sampling feathers or egg membranes.
However, note that chelex extractions are not recommended unless it is
anticipated there will be no long-term use of the extracted DNA.}

**[Feathers from museum skins]{.underline}.** *(Feathers/1 Vial/Tissue
Preservation Buffer/Room Temperature)*

Success in extracting DNA from museum skins is variable, depending upon
the way the skin was prepared. We have developed protocols for
extracting from samples prepared using a number of preservatives (and
combinations), including gasoline, arsenic and borax. We generally get
better extractions from feathers plucked from museum skins (along with
skin at the base of the feather), rather than from snips of skin alone.
This may be due to inhibitors from preservatives used on the skins.

To minimize damage to museum skins, try to collect from areas that are
less noticeable. Sampling can often be more easily done along suture
lines, such as in the area of the cloaca. Feathers from the wing area
(such as the marginal coverts) usually yield good DNA (perhaps because
preservatives were used less often on the wings), but sampling from this
area is difficult to do without affecting the integrity of the skin.
Please abide by the instructions of the curators. We like to have at
least 5 feathers and associated skin if possible.

Pluck feathers and associated skin from a small area. It helps sometimes
to use forceps or tweezers. Take care to support the skin with one hand
while gently pulling the sample; this will help to keep the skin from
ripping. Place each sample in a sampling envelope and record museum
numbering system, and species, sex, age, date and collection location.
Often the museums will have much of this information on databanks. Keep
the envelope dry.

We strongly suggest that latex gloves be used when sampling, and that
instruments be cleaned with 10% bleach between sampling. This prevents
between-sample contamination and protects the collector from infectious
diseases and any preservatives that may have been used in the skin's
preparation.

If we are preparing skins, we always collect tissue samples from the
carcass. We collect heart, breast muscle, and blood as it pools around
the heart, and store each in separate tissue preservation buffer vials
(see protocols below, and separate multiple tissue sampling protocol).

See Mundy et al. (1997) for additional information about collecting from
museum skins.

# Fin Tissues. *(Tissue/1 Vial/Ethanol/Room Temperature) or (Tissue/1 Container/Dry/Room Temperature)*

Fifty fin samples for population genetic analysis and three or four
reference samples (whole fish) for phylogenetic analysis need to be
collected from each location. There are two methods for sample
collection: dry or in vials with 100% ethanol (EtOH). The *preferred
method of collection* is to store the tissue in a vial with *100%
ethanol (EtOH).* (If this is not possible, the following dry method can
be used.)

Use clean scissors or a clean scalpel blade to cut a small piece of
tissue from one of the fins of the live fish. Tissue size should be
approximately 5 mm2 ( about the size of this block ). A wedge from the
upper or lower lobe of the tail fin works fine. Because adipose fins
contain a lot of complex lipids, they are not an easy target for DNA
extraction, although some DNA can be extracted from this tissue. Eroded
fins from dead salmon carcasses are highly degraded, and DNA is usually
not readily extracted from such tissue. A well-dried 5 cm2 piece of skin
tissue works best under these conditions.

The date of collection, fish species and stock, type of collection
method, and fish length, sex, and age (YOY, juvenile, adult will
suffice) should be collected with each fin where possible.

If samples are to be sent through the mail, ethanol should be drained
from the samples immediately prior to mailing; the samples will be
rehydrated upon receipt at the Molecular Ecology Laboratory.

Once the samples have been collected, please contact Talia Wiacek,
Biological Science Technician, (907) 786-3494, Talia_Wiacek@usgs.gov.

***[Dry Sample Collection (Alternative)]{.underline}.*** For the dry
method, whirl-pack bags, cryo-tubes, or scale envelopes lined with high
quality filter paper work well.

Either in the field after collection, or in the office immediately upon
return from the field, samples should by air-dried on filter paper or
paper towels until all mucus and moisture in the fin has evaporated and
the fin feels dry to the touch. Sun drying in the field works best and
can be done quickly. Drying fins inside usually takes 18-24 hours at
room temperature. Fungus and bacteria immediately invade the fins upon
collection and these factors break down the cell walls of the tissue and
the DNA exudes into the surrounding medium, making DNA extraction in the
lab difficult, if not impossible. DNA from moist-stored fins are often
OK for up to 6-8 hours (it depends on the original condition and size of
fin clip), but samples are best when packed on ice if drying is to be
delayed for over 4 hours.

Dried fin clips should be repackaged separately (make sure the baggy or
envelope is dry as well) and attached to field notes for shipment. Dry
samples can be sent surface mail without special packaging.

**[Hair and Hard Tissue]{.underline}** *(Hair, Bone, or Teeth/1
Envelope/Dry/Room Temperature)*

Hairs and hard tissue samples, such as bone or teeth, should be kept as
dry as possible. DNA is present only in hair follicles, so hairs without
follicles are not useful for genetic analyses. DNA is extracted from
tooth pulp, so the whole tooth is preferred. These can be stored in
containers or envelopes.

# Muscle (*Tissue/1 Vial/Tissue Preservation Buffer/Room Temperature)*

Muscle tissue samples are the preferred samples for work that includes
mtDNA analyses along with nuclear DNA analyses, particularly for birds.
Among muscle tissue samples, heart is the most preferred for birds,
since the mtDNA yield is very high relative to nuclear yield. DNA can
also be extracted from tongue, skin, hair, teeth and bone. Soft tissue
samples can be stored at room temperature in the field in the tissue
preservation buffer. Any muscle or skin tissue will work and can be
stored in this buffer solution. **Please make sure that the storage
buffer completely covers the tissue sample**. Also, make sure to clean
instruments between sampling different birds to prevent
cross-contamination, using a 10% bleach solution **followed by a water
rinse**. A sample about the size of a pencil eraser is all that is
needed, but make sure the sample is entirely submersed in the buffer.

# Scat *(Scat/1 Vial/EtOH/Room Temperature) or (Scat/1 Vial/Silica Beads/Room Temperature)*

Host DNA is very difficult to obtain from scat samples. Because of the
low amounts of host DNA compared to bacterial and diet sources of DNA,
extreme caution must be used to prevent contamination of one scat sample
with scat from another individual. Therefore, we provide gloves, and
tongue depressors with each sampling vial to be used only for one vial
then discarded. We provide two different types of preservative for scat
samples: (1) Liquid ethanol in a 50 ml tube, or (2) Silica beads/gel in
a 50 ml tube. Unpublished research from our laboratory and others
suggest that ethanol is superior to silica beads/gel for preserving scat
samples for DNA analysis.

Regardless of which preservative you have, use a new pair of gloves and
a new tongue depressor for each sample handled. If you are directed to
aliquot one scat sample between the two preservative types, you do not
have to change gloves and tongue depressors. Try to place an amount of
scat approximately the size of a golf ball into the collection tube. Do
not fill the tube; it is important to leave enough space for the sample
and preservative to mix (easier done with the liquid ethanol than the
silica beads). Samples are okay left at ambient temperature, but should
be kept away from heat and out of sunlight for a few days. It is best to
return them to the lab and freeze them as soon as possible. Unpublished
data from several laboratories, including ours, indicate DNA yields
decline dramatically in samples over about a week old, regardless of
collection method. Scat samples preserved within 24 hours of defecation
yield the highest amount of host DNA.

### 

### Chemical Descriptions and Hazards

(For more specific details on each chemical ingredient, see the attached
Material Safety Data Sheets.)

# Blood Buffer

**Tris/HCL**. May cause irritation to skin and mucous membranes on
contact. Wash contacted area with plenty of water and contact physician
if irritation persists. Ingestion of large doses may cause interior
irritation, nausea, weakness and collapse. If ingested, drink copious
amounts of water and call a physician.

**EDTA**. May cause irritation to skin and mucous membranes on contact.
Wash contacted area with plenty of water and contact physician if
irritation persists. If ingested, drink copious amounts of water and
call a physician

**NaCl (Sodium chloride)**. May cause irritation to skin and mucous
membranes on contact. Wash contacted area with plenty of water and
contact physician if irritation persists. If ingested, drink copious
amounts of water and call a physician

**SDS (Sodium dodecyl sulfate).** May cause irritation to skin and
mucous membranes on contact. Wash contacted area with plenty of water
and contact physician if irritation persists. If ingested, do not induce
vomiting. Drink copious amounts of water and call a physician.

## Tissue Buffer

**Tris/HCL**. See above.

**EDTA**. See above.

**NaCl (Sodium chloride)**. See above.

**N Lauroyl sarcosine.** May cause irritation to skin and mucous
membranes on contact. Wash contacted area with plenty of water and
contact physician if irritation persists. If ingested, do not induce
vomiting. Drink copious amounts of water and call a physician.

**[Urea]{.underline}**. May cause irritation to skin or eyes. Wash
contacted area with plenty of water and contact physician if irritation
persists. If ingested, do not induce vomiting. Drink copious amounts of
water and call a physician.

**[Silica Beads]{.underline}.** Silica is an inhalation hazard. Do not
breath silica dust or leave in open areas.

# Ethanol. Ethanol is highly flammable; do not place near an open flame. High vapor concentrations or consumption can cause narcotic effects. Do not breath vapors or consume this ethanol.

**Additional Sampling Information**

Mundy, N. I., P. Unitt, and D. S. Woodruff. 1997. Skin from feet of
museum specimens as a non-destructive source of DNA for avian
genotyping. *The Auk* 114(1):126-129.

Pearce, J. M., R. L. Fields, and K. T. Scribner. 1997. Nest materials as
a source of genetic data for avian ecological studies. *J. Field
Ornithol.*, 68(3):471-481.