FISH/BIO 340: Genetics and Molecular Ecology

Autumn 2014
Lab 1: DNA Extraction & Pipetting
Please print off, read through (!) and bring a copy with you to the first lab in FTR 113
Answer the pre-lab questions on Canvas, before the start of your lab section
(https://canvas.uw.edu/)
** Dont forget to write the Purpose and write/paste the Methods in your notebook for the
pre-lab notebook check.**
Schedule:
Part A - Pipetting exercise
Part B - Begin DNA extraction
Part C - Brief discussion of maintaining a lab notebook
Part D - Bat morphology and identification
Part E - Complete DNA extraction

Purpose:
In this lab you will perform the first step of our quarter-long research project: extraction of DNA
from bat tissues. You will use this DNA for the rest of the lab project to ultimately identify our
mystery bats. Additionally, you will learn the essential technique of micropipetting small
volumes and maintaining a lab notebook. Finally, we will attempt to identify our bats using
morphology.

Part A: Pipetting
Proficient pipetting is probably the most crucial part of
this class, for three reasons:
Accurate pipetting is crucial to the success of
molecular projects. Mistakes during pipetting may
cause your experiments (e.g. PCR) to fail or to be
irreproducible, and thus cause long delays and
considerable expense
Pipettes are delicate pieces of equipment with high
accuracy, which can easily be knocked off their
calibration. Furthermore, they are expensive each
pipette costs about US$ 250 -300.
Pipettes are a common source of contamination in
molecular work.
You may have some experience with pipettes, but a
refresher on pipetting is probably quite useful.
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Volume Adjustment

Red/black indicates the decimal point.
Never
Never rotate volume adjustor beyond the upper limit
Changes calibration
Never use without a tip.
Liquid will get into piston corrosion and contamination.
Never lay down or turn upside down a pipette with filled tip.
Liquid could run back into the piston, and damage/contaminate it.
Use pipette holders provided
Never let plunger snap back after withdrawing or ejecting fluid
Damages piston

Proper Use of a Pipette
Use the correct pipette. Never adjust the volume beyond the maximum setting, but also do
not use pipettes that are too large. Accuracy and precision drop rapidly towards the lower
limit of settings.
Pipette Top Volumes (l) Tips
P-1000 Blue 100 1000 Blue
P-200 Yellow 20 200 White, tall
P-100 Yellow 10 100 White, tall
P-20 Yellow 2 20 White, tall
P-10 White 0.1 10 White, small
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Adjust volume by turning the volume adjustment knob or the plunger. Be sure to locate
the decimal point correctly when reading the volume setting (see above). Always dial
DOWN to the desired volume to avoid mechanical backlash affecting accuracy.
Firmly seat the proper-sized tip on end of the pipette. If the fit is loose, you will draw up
less volume than intended, and the liquid will drip from the tip during use.
When withdrawing or expelling fluid, always hold the tube firmly between thumb and
forefinger. Hold close to eye level to observe change in fluid level in tip. Do not pipet
with the tube in rack or when someone else is holding the tube.
The pipettes have a two-stop position plunger. Depressing to the first stop measures the
desired volume. Depressing to the second stop introduces an additional volume of air to
blow out any remaining fluid from the tip.
To withdraw fluid from tube.
Hold the pipette almost vertically (< 20
o
from vertical)
Depress plunger to first stop and hold. Dip tip 2-4 mm into the fluid. Do not push
down to the bottom of the vial, otherwise the tip is blocked and you draw less liquid
than intended.
Gently release thumb. If you release quickly, you will create aerosols (small droplets)
which will contaminate the pipette.
Wait for a second or so, to confirm that all the liquid has been taken up.
Slide pipette tip out along wall of tube to dislodge remaining droplets adhering to the
outside of the tip.
Check that there is no air space at the very end of the tip.
Learn the approximate levels that particular volumes fill the tip this will allow you
to check your pipetting visually.
To expel sample into tube
Touch tip to wall of tube
Slowly depress plunger to first, and then to the second stop to expel fluid.
While keeping the plunger at the second stop, slide the tip out of the fluid, along the
tube wall and out of the tube.
Eject the tip into the tip waste container by pressing the tip ejector button.
Prevention of cross contamination
Use a fresh tip each time
Do not touch the tube with the pipette, only with the tip
If you suspect pipette contamination, wipe with ethanol on the outside. If the inside is
contaminated, talk to your TA or Lorenz.
Draw up liquid slowly to prevent the formation of aerosols
For specific applications, use pipette tips with filters
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Pipetting exercise (to be completed in ~ 20 mins)
We will be using colored water in the exercise so you can easily visualize what is happening.
Be careful to use the correct pipette:
For volumes less than 10l, use a P10 (white pipette tips)
For volumes between 10 and 200l, use the P200 (larger, white or yellow tips)
For volumes between 100 and 1000l, use the P1000 (blue pipette tips)

In addition to completing the pipetting exercises, please also answer the following questions in
your lab notebook:
1. Add 279 l to a 1.5mL flip-top tube. Which pipette(s) do you want to use?
2. Add 1,120 l to the same tube. Which pipette(s) do you want to use?
3. Remove 502 l from the same tube. Which pipette do you want to use?
4. Add 84 l to the tube. Which pipette do you want to use?
5. Add 507 l to the tube. Which pipette do you want to use?
6. Add 4.3 l to the tube. Which pipette do you want to use?
7. Add 7.7 l to the tube. Which pipette do you want to use?
8. Record qualitative observations regarding how accurately you were at pipetting water. Be
sure to note any problems you had with pipetting (i.e. air in the tip; were not able to expel
all the liquid, excess water on the outside of the tube, etc.). How close are you to the
1.5ml mark on the tube? If you are above or below the line, what are some possible
sources of error?

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Part B: Begin DNA extraction using Qiagen DNeasy Extraction Kit
A note on tissue collection and preservation
Tissue breaks down rapidly after an organism dies. While it is possible to use DNA from
very old cells (fossils or tissue frozen in permafrost, for example), the DNA is usually severely
degraded if the tissue is not preserved soon after the death of the organism. This degradation
means that the long strands of DNA have been broken into very small pieces (between 1 and 200
base pairs), which severely limits the usefulness of the DNA.
To prevent degradation, tissue is preserved or fixed immediately when it is collected in
the field. In molecular ecology, tissue is usually either frozen in liquid nitrogen or on dry ice,
or it is preserved in 90-100% ethanol. Both these methods essentially dehydrate the tissue and
therefore prevent any enzymatic activity degrading DNA. Unfortunately, most museum
specimens are usually preserved in formaldehyde, which renders DNA useless for most molecular
genetic investigations. However, our bats have been frozen, which usually preserves DNA fairly
well.
Cell Lysis
1. Put on gloves
2. Each student will obtain a thawed bat from your TA. Fill out a bat ID tag (pictured below)
for each bat and the Record in your lab notebook the number of the bat individual
(the bat ID number can be located were XX is in the picture below). You can determine
the sex of your bat by using the presence or absence of a phallus.
3.





4. Record in your lab notebook which tissue and approximately how much tissue you
remove from your bat for DNA extraction. Your TA will provide more instructions on
which parts of the bat to use in lab.
5. Remember to label all tubes with your group number, initials, and date, as well as the bat
ID #. Use the top of the tube for quick reference information (your group number, initials,
bat) and the side of the tube for details (date, group, your initials).
6. After you have removed your bat tissue and placed it in a tube, put your bat along with the
ID tag in plastic bag and set aside. You will take morphological measurements later in
lab; however it is important avoid mixing up bats once you have started the DNA
extraction. This ensures the DNA sample can be linked with the bat

Bat # XX Date:
DNA Extracted By:
Lab Section:
Bat Sex:
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Part C: Lab notebook
General instructions:
Think about your lab notebook as a record of your work. It should be sufficiently detailed
that you (or any other competent person) could go back and exactly repeat your
experiment (including errors if so desired).
Use a hardback bound notebook (not spiral-bound), so you dont loose sheets.
The first page should be the title page. Include your name, lab section, and contact
information.
Number the pages
Create a table of contents
List all materials, samples and reagents that you are using
Remember to keep exact record of your errors: some of these errors lead to important new
discoveries, but only if you can repeat them (e.g. the discovery of Penicillin).
Start each lab on a fresh page, so you can find it again.
Say exactly what was done, by whom, and when.
Black ballpoint pen is recommended.
Cross out mistakes with a single line, like this.
Take up as much space as you need. Use your biggest, best handwriting other people
should be able to read it.
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Create a table of abbreviations if you use them.
Before lab:
For each lab, start on a fresh page and write: the title, purpose, and date of the experiment.
Write down the methods to be used, including recipes and equipment. (Use this to really
become familiar with the protocol for the lab.) You may type out methods or tape/paste
copies of the methods from your lab handout, however highlight or underline important
sections (i.e. reagent and volume) and make notes as you complete the steps in lab
We will inspect your notebooks for this pre-lab work before you can begin the lab.
During lab:
Record your observations and data. Do this honestly and as things happen. Remember to
cross out mistakes with a single line.
Write out any calculations clearly, including all steps and units.
Take notes during the lab as much as possible. Only write notes after lab if this is
unavoidable, and clearly mark such sections as written after lab.
We will provide specific advice on the information that should be in your lab notebook in
each lab.
We will discuss lab notebooks more extensively in the lab. More extensive guidelines (down to
the make of pen you should use) can be found at
http://www.swarthmore.edu/NatSci/cpurrin1/notebookadvice.htm

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Part D: Bat Identification Using Morphology
First, we will test whether we can identify the bats morphologically. There has been great interest
in bat conservation, so species are well described. You will take measurements and counts and
compare them to species description. Be careful in your measurements and counts as the
differences between species are quite small.
Fill in the table below in your lab notebook with the measurements and counts for your
groups bats. Each group member should use these measurements and counts along with the
tables in the appendix to determine possible species identifications for their unknown bat.

Bat 1 Bat 2 Bat 3 Bat 4 Bat 5
Group Members Name
Bat ID #
Total length (rostrum to tail)
(mm)

Tail length (mm)
Wingspan (mm)
Right foot (mm)
Right ear (mm)
Tragus (mm)
Forearm (mm)
Keel (+/-)
Sex

Age (Juvenile or Adult)
(degree of ossification in the
phalangeal joint)

Weight (g)
Possible Species:



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Bat Anatomy:




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*Key Reference: Nagorsen (2002) An Identification Manual to the Small
Mammals of British Columbia
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Part E: DNA Extraction
The purpose of the second part of lab is to provide you with a tangible way of thinking
about DNA as well as to introduce you to some of the basic lab techniques used in genetics and
molecular ecology. The first step of the overall lab project will be to extract genomic DNA from
bat tissue.















As shown above, there are a variety of methods available for extracting DNA from cells, but they
all have similar components. First the DNA must be released from the cell (Cell lysis; animal,
plant, bacterial, etc). Typically, a solution containing detergent (soap) of some sort (e.g. SDS or
Sodium Laurel Sulfate a common ingredient in shampoo) is used to lyse cell membranes and
break up proteins. Next, an enzyme (e.g. Proteinase K) is added to digest cellular proteins. DNA
is then separated from all the cellular debris (Protein removal); this is the point where most
extraction protocols differ. After all proteins and cellular debris have been removed, the DNA is
precipitated out of solution usually by adding alcohol (DNA precipitation). DNA is then
collected by centrifugation. Once the DNA is dried to remove the alcohol, it is resuspended in a
buffer that helps preserve the DNA.
These days, commercially available kits make DNA extraction much easier and faster. They work
in a similar way to the above, first digesting the tissue and then separating proteins and DNA.
However, in these kits, the DNA is bound to a special membrane after the digestion and
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contaminating substances are washed away. After the wash, the DNA is eluted (separated) from
the membrane and collected in a tube for storage.

We will use the Qiagen DNeasy Extraction Kit (take a note of that for your lab notebook
and report)
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Complete DNA Extraction




NOTE: Before transferring mixture from step 3 into the mini spin column
in step 4 label the mini spin column with the sample ID








NOTE: Label the microcentrifuge tube with your name, group number,
and sample ID.
8. Bring your tube DNA extraction to the TA for storage at 4C
until the next lab.


100
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Pre-lab Questions
(Complete on Canvas before the start of lab)

1. Which enzyme will we use to digest tissue for DNA extraction?
2. Why are some numbers in red and some in black on our Rainin pipettes?
3. Why is a lab notebook important?
4. Why is accurate pipetting so important in molecular projects?
5. What is the purpose of the DNeasy Mini spin columns?