Class Set
Techniques Lab A: Manipulating Small Volumes
Background
A forensic scientist removes a microscopic amount of DNA from a dime-sized drop of blood left
at the scene of a crime. A cystic fibrosis patient inhales a fine mist containing "good" copies of a
gene he did not inherit. When the criminologist and the genetic engineer perform laboratory
procedures involving tiny amounts of DNA and other chemical solutions, each uses an
instrument known as a micropipette.
A micropipette is a kind of fancy eyedropper -- one that comes in many different models and
volume ranges. But while an eyedropper dispenses drops, micropipettes transfer microliters of
fluid. In the metric system, the basic unit of volume is the liter ("L" or "l"). If you put the prefix
"milli-" which means "one-thousandth", in front of liter, you are referring to one thousandth of a
liter, or one milliliter ("mL" or "ml"). "Micro-" is a prefix in the metric system which means "onemillionth" of the base unit. Therefore, one microliter ("µL" or "µl") is one-millionth of a liter. It may
be easier for you to picture one milliliter (mL or ml) of water. If you mentally divide that milliliter
of water into 1000 tiny equal-sized volumes, each volume is one microliter.
Purpose
This laboratory activity introduces micropipetting technique. As with all fine motor skills, learning
how to use a micropipette takes practice and determination. You will be rewarded with excellent
DNA laboratory results in this and future labs.
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PART 1--Directions for Using Micropipettes
CAUTIONS:
Set pipette volume only within the range specified for that
micropipette. Do not attempt to set a volume beyond the pipette's
minimum or maximum values.
When using a micropipette, first apply a tip. Forgetting to do this
would cause liquid to be sucked into the nose cone. Since a
micropipette works by air displacement, its internal mechanism
must remain dry.
Always keep a micropipette in a vertical position when there is fluid
in the tip. Do not allow liquid to accidentally run back into the nose
cone.
Use your thumb to control the speed at which the plunger rises
after taking up or ejecting fluid. Releasing the plunger too abruptly
will cause liquid to pop up into the nose cone and damage the
piston.
Setting and Preparing the Micropipette
1. Check that you have the right micropipette. There may be three sizes in the lab -- a "P-20"
(for 2 to 20 µL), a "P-200" (for 20-200 µL), and a "P-1000" (for 200-1000 µL).
2. Dial the desired volume (start with 4 µL).
3. Push the end of the pipette into the proper-size tip. The yellow tips are for P-20's and P-200's.
How to Take Up Sample with a Micropipette
4. Before picking up the micropipette, open the cap or lid of the tube from which you are taking
fluid (or, have your lab partner do this and hand the tube to you).
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5. Hold the micropipette in one hand, almost vertical; hold the tube
in your other hand. Both should be almost at eye-level.
NOTE: the person holding the micropipette is also the person who
should be holding the tube!
6. Before lowering the tip into the liquid, push down the plunger to
the first stop and hold in this position.(DO NOT GO PAST THE
FIRST STOP OR YOU WILL GET AN INCORRECT VOLUME),
then...
7.
... dip the tip into the solution to be pipetted.
8. Draw fluid into the tip by slowly releasing the plunger. (Cap and
put down the tube with the liquid or hand it to your partner.)
How to Expel a Sample From the Micropipette
9. With your other hand (or, have your lab partner do this), open the cap or lid of the tube into
which you are ejecting the fluid.
10. Hold the micropipette in one hand, almost vertical; hold the tube
in your other hand. Both should be about at eye-level.
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11. Touch the micropipette tip to the inside wall of the reaction tube
into which you want to expel the sample. This creates a tiny
surface-tension effect which helps coax fluid out of the tip.
12. Slowly push down the plunger of the micropipette to the first
stop. Then, continue to the second stop to expel the last bit of fluid,
and hold the plunger in this position.
13. Slowly remove the pipette out of the tube, keeping the plunger pushed down to avoid
sucking any liquid back into the tip.
14. Always change tips for each new reagent you need to pipette. To eject a tip, depress the
large blue button on the top of the micropipette.
Techniques Lab A: Manipulating Small Volumes
ACTIVITY--Practice with the Micropipette
I.
Practicing with a P-20
1.
2.
Label two empty reaction tubes A and B with a permanent ink marker, if not
already labeled.
Add the amounts of solutions I, II, and III to tubes A and B as shown in the table
below.
Solution Volume in Each Tube
Reaction Solution Solution Solution
Tube
I
II
III
3.
A
4 µL
5 µL
2 µL
B
6.5 µL
2.5 µL
2 µL
Total in
Tube
Spin tubes A and B in the microcentrifuge for a few seconds to pool the solutions.
Refer to the Centrifuge Instructions on the last page.
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4. Add up the total volume of liquid in Tube A. AS A CHECK
OF YOUR TECHNIQUE, set the micropipette to that volume
and withdraw all of the liquid in tube A. The contents should
just fill the tip -- no air space at the bottom of the tip; no
leftover liquid in the tube. Discard liquid and tip into waste
beaker.
5. How much should be in tube B? __________ Check your
technique by setting the pipette to the correct volume and
withdraw all (hopefully) the solution in tube B.
II.
Practicing with a P-200 – If you have time while other groups are finishing, do this!
1.
Label an empty reaction tube C, if not already labeled.
2.
Add the amounts of solutions I, II and III to tube C as shown in the table below.
Reaction Solution Solution Solution
Total in Tube
Tube
I
II
III
C
3.
20 µL
40 µL
100 µL
Spin tube C for 1-2 sec. (Be sure to balance your tube.)
4. Check the accuracy of your micropipeting technique with the P-200. Set to
________ µL and withdraw the contents of tube C.

Assessment X
1. Use one of your colored water reaction tubes.
2. One at a time, each student in your lab group of 4 should withdraw 9 µL of water
and expel the 9 µL as one drop on a piece of waxed paper. When each student
has had a turn, compare the four drops. Are the drops the same size? If yes,
great! Continue to step 3.
If not, why not? Try again, carefully coaching each other.

Assessment Z
1. Obtain a tube with 10 µL of glycerol.
2. Each student in a group of 4 should withdraw 2 µL from the tube and place it on a
piece of waxed paper as separate drops. Compare drop sizes
3. When everyone in the group is finished, call the teacher over to check your results
by withdrawing the last 2 µL.
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Upon completion of this lab





Dispose of all tips and used reaction tubes in waste container.
Dispose of designated materials as outlined by your instructor.
Leave equipment as you found it.
Check that your work station is in order.
Wash your hands.
Techniques Lab A: Manipulating Small Volumes
PART 2--Centrifuge Instructions
1. When you are adding several reagents to one tube, release each drop of reagent on the
inside wall of the tube near the bottom.
2. Tightly close the caps on all the tubes to be placed in the microcentrifuge (also called
microfuge).
3. The microfuge rotor must always be balanced - you cannot, for example, insert one tube
into a microfuge. Spinning in an unbalanced arrangement like this would damage the
motor of the instrument.
4. The amount of liquid in the tubes should be similar; otherwise the rotor will spin unevenly
(like wet towels spinning out of balance in a washing machine). You can always prepare
a "blank" tube with the appropriate volume of liquid with which to balance a single tube.
Samples of balanced rotor configurations:
Question: What do you do if you only have 5 tubes?
You must put in one extra (a blank) tube blank with equal volume.
5. After you have replaced the plastic top and secured the lid of the microfuge, give the
tubes a 1-2 second pulse. This will mix and pool all the reagents into a droplet in the
bottom of each tube.
NOTE: REVIEW SECTIONS OF INSTRUCTIONS AS NEEDED AS YOU PROCEED TO
PRACTICE MICROPIPETTING
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