How to sample dung for genetic analyses!
Dung is great source of information. Today, we not only get information on diet, health, and breeding information from dung, but now we can also extract DNA from an appropriately sampled pile of dung. This facilitates sampling for large and elusive animals, reduces the stress and costs associated with capturing the animals. Here we describe in a few simple steps on how to collect, and preserve dung for genetic analysis.
First Step: Before you go out to the field!
Before going out to the field to collect dung, you should make sure you are taking the following:
1. A notebook to keep a record of each sample.
2. Parafilm to seal the sampling tubes. Parafilm is usually easy to find, any lab supplier should have it, and any molecular biology lab will have it, too.
3. A preserving agent of some sort to preserve your samples until you get a chance to extract the DNA. You usually have a choice of preserving agents, and should use what is most appropriate for your field condition, and budget, look below for a short list of options.
4. A GPS unit to obtain a geographic position for each sample.
5. A sharpie, or a permanent marker of some kind, to write on the sampling tube, and a pencil to write in the notebook (pencil is better than pen, pencils don't smudge!). If you decide to use alcohol as your preserving agent, you might consider getting yourself an alcohol safe permanent pen.
6. Sampling tubes. I usually use a 5ml plastic tube, with a screw top lid. I also usually prepare 10-15 tubes with roughly 4ml of preserving agent before i go to the field. I get my tubes from VWR.
Preserving agents act by dissecating the
sample. Water is the medium through which DNA degrading
enzymes act, furthermore, DNA molecules suffer hydrolysis.
So, you want to remove as much water as possible from the
medium. Some good preserving agents are:
a. RNAlater is a preserving liquid, non-toxic and
non-flammable designed specifically to preserve RNA and DNA
at room temperature. Usually, DNA extracted from the same
sample preserved with RNAlater than preserved with alcohol,
or dry. You can read more about RNAlater at the Ambion website.
b. Ethanol is another good way to preserver your
samples, and certainly more accessible. However, because it
is flammable, it is sometimes more difficult to transport.
Use 4ml or so of ethanol at a concentration from 70-95% to
get good results. You can keep the samples at room
temperature, but 4 oC it will last longer. Other types are
not recommended, not only because of their toxicity to
humans, but also because they interfere with the DNA
extraction procedure.
c. Silica gel (the same used to keep humidity away
from camera equipments) is also a great preserving agent,
easy to transport, non-toxic and non-flammable. You start
by filling up the tube with 3-4ml of the little silica gel
beads, then cover it with a small piece of paper towel or
filter paper, so there isn't direct contact between the gel
and the sample. Then, place roughly 2cm² of sample on top
of the paper towel. Silica gel can usually be purchased in
any camera specialty store or lab supplier.
Second Step: What is a good sample?
Find fresh dung, the fresher the better. Although, DNA has also been successfully extracted from older (i.e. dry) dung, with successful amplification of PCR products.
Third Step: What should I sample?
As dung passes through the intestines of the animal, cells from the intestine's wall are carried with it. Consequently, the best sample for genetics is the surface of a fecal matter. That is where the greatest concentration of the individual's DNA will be found. A good amount to collect is about 2-3 cm².
Collection should be done using available dried twigs that are lying around the site. Use new twigs for each new pile of dung you find. Use the twig to scrapped off a section from the surface, and put it into your collection tube. Avoid contaminating the sample with your own DNA by touching it with your bare hands; or, with the DNA from other samples, by using the same twigs. If you decide to use gloves, use a fresh pair for each new pile of dung. If you decide to use tweezers, make sure to take alcohol, matches and plenty of paper towels with you, because you want to clean and sterilize the tweezers in between sampling bouts.
Fourth Step: Avoid Spills!
Once the sample is in the tube, and the top is back on, it is good to seal the top with parafilm. You can take pre-prepared parafilm strips to the field, or take a knife or pair of scissors to cut them on the spot. You need roughly a 2cm wide strip of parafilm. Put one tip on the tube, so that half the strip in on the lid and the other half on the tube.
With one hand holding that tip, pull the parafilm tightly around. So, that the tube is effectively sealed. Finally give it a good shake to mix the sample will with the preserving agent (if is it a liquid agent)
Fifth Step: Mark the Spot!
It is essential to always take a GPS measurement for each sample you take. The spatial location can be useful, and many times, essential in analyzing and interpreting patterns both for studies focusing on population genetics, as well as in ecological studies that are using molecular techniques simply as a tool.
Sixth Step: Cover up!
Once you are done collecting your sample, you might consider throwing leaves, and generally making a mess of the pile you have just collected from. This will avoid you sampling twice from the same sample.