Use of Micropipettors
Micropipettors are instruments used to accurately transfer small volumes (1 l to 1 ml) of solution. Because of
their accuracy, ease of use, and convenience in sterile techniques they are a nearly universal lab tool. In this lab
you will learn how to properly use this instrument. Use the terms and the illustration below to become familiar
with the parts of a micropipettor:
TERMS
Barrel or shaft - the working end of the
micropipettor; seat a disposable tip on
the lower end of the barrel before each
use.
Plunger – press the plunger to withdraw
liquid. Release it to expel liquid.
Tip eject button - press to remove a tip
from the barrel without direct handling of
the disposable tip.
Volume readout - displays the volume
the micropipettor is currently set to
deliver.
Volume adjustment knob - rotate to
change the digital volume setting.
Image adapted from: http://www.di.uq.edu.au/sparqmicropipette
On the lab bench, you will find four micropipettors; each is appropriate for a specific volume range. If you look
at the dot on the plunger of each micropipettor you will see a number that represents the maximum volume, in
microliters (l), that can be transferred by that micropipettor. The minimum volume appropriate for each
micropipettor is typically ten percent of the maximum. The dot on the plunger is also color-coded and often
matches the color of the disposable tips used with that micropipettor. The table below shows the volume range,
expected accuracy, and the appropriate tips for the micropipettors that you will be using.
Micropipettor
P1000
P200
P20
P10
Volume Range (l)
100-1000
20-200
1-20
1-10
Disposable Tip
Accuracy (l)
 10.0
 1.0
 0.5
± 0.5
Blue
Yellow
Yellow
Clear
Adapted from: DNA Science, 2003 by Cold Spring Harbor Laboratory Press, purchased from Carolina Biological.
In most experiments, accuracy is important when transferring small volumes of liquid with a micropipettor. A
researcher needs to be sure that he or she is transferring the volume desired with a reasonable degree of
accuracy. The researcher can be confident of this provided two conditions are satisfied. First, the micropipettor
has been calibrated and tested for accuracy (this is usually done on a yearly or semi-yearly basis). Second, the
researcher must be using the micropipettor properly.
Part 1: Familiarization with the micropipettor
Materials: set of micropipettors
Pick up a P20 and set the digital volume setting for 5 l (reading down, the setting should be "050'"). Push the
plunger down and notice that at some point the plunger becomes more resistant and requires more effort to
push further. This point is called the first stop. Notice that the plunger can be pushed well beyond the first stop
until it reaches "the second stop". Be sure you can feel the first stop point and notice how far the plunger travels
before reaching this point. Reset the micropipettor to 20 l ("200"). Again push the plunger to the first stop. You
should notice now that the plunger travels further to reach the first stop than it did when the micropipettor was
set for 5 I. Examine the P10, P200, and P1000 setting windows as well. The table below shows how the volume
display looks for each micropipettor when it is set at its maximum volume.
Micropipettor
P1000
P200
P20
P10
Maximum Setting
1
0
0
2
0
0
2
0
0
1
0
0
Look at the diagram below, which shows how to read the volume of a micropipettor.
Image adapted from: http://faculty.buffalostate.edu/wadswogj/courses/BIO211%20Page/Resources/micropipetting%20lab.pdf
Practice determining the volume on the diagram below. Write the correct volume for each micropipette.
P10
P20
P200
P1000
0
5
6
1
3
2
1
8
8
0
4
7
volume
volume
volume
volume
Take up sample with micropipette:
1. Set pipet volume by rotating the volume adjustment knob. Note: the volume setting should never be
adjusted above the maximum volume specified for a particular micropipettor. Remember that the
maximum volume is the volume shown on the plunger.
2. Seat a disposable tip on the micropipettor by placing the end of the barrel into a tip. To avoid
contamination, use a new tip for each transfer.
3. Depress plunger to first stop and hold at that spot.
4. Place tip into the liquid to be pipetted.
5. Slowly release plunger to draw up liquid. Do not release too quickly or you will create air bubbles.
6. Wipe any excess droplets on side of container, but do not touch the tip.
Release sample from micropipette:
1. Put tip into tube and touch the inner wall of tube with tip.
2. Press plunger down to the second stop and hold it there.
3. Pull micropipette out of tube and release plunger once it is out of tube. This prevents you from
inadvertently drawing in liquid.
4. Eject the used tip. Place the tip over the appropriate waste container and press the tip ejector button. If
the tip is difficult to eject it is likely that you are jamming the tips onto the micropipettor harder than
necessary.
Part 2: Transferring small volumes with a micropipettor
Materials:
Piece of blotting paper
2X Loading dye
P20 and rack of yellow tips
P10 and rack of clear tips
Obtain a 5 x 5-inch piece of blotting paper and a microcentrifuge tube containing 2X loading dye. Follow the
protocol above and use a P10 to spot the following volumes of dye directly onto the paper in a linear order: 1 l,
3 l, 5 l, and 7 l. Repeat using the P20 for: 10 l, 15 l, and 20 l. Repeat in a second row directly below the
first. Compare your first and second rows to check your consistency. Compare the spots you made with those of
other students. If the spots do not look like those of the instructor, try to determine the cause.
Part 3: Transferring large volumes with a micropipettor
This exercise simulates parts of a bacterial transformation or plasmid preparation for which a 100 - 1000 l
micropipettor is used. It is far easier to mismeasure when using a large-volume micropipettor. If the plunger is
not released slowly, an air bubble may form or solution may be drawn into the piston.
Materials:
2 x 1.5 ml microcentrifuge tubes
Solutions I - IV
P1000 and rack of blue tips
1. Use a permanent marker to label two 1.5 ml reaction tubes E and F.
2. Use matrix below as a checklist while adding solutions to each reaction tube.
Tube
E
F
Solution I
100 l
150 l
Solution II
200 l
250 l
Solution III
150 l
350 l
Solution IV
550 l
250 l
3. Set micropipettor to add appropriate volumes of Solutions I-IV to tubes E and F. Follow the same
procedure as for small-volume pipettor.
4. A total of 1000 l of reactants was added to each tube. To check that your measurements were
accurate, set the micropipettor to 1000 l and carefully withdraw solution from each tube.
a. Is the tip just filled?
or
b. Is a small volume of fluid left in the tube?
or
c. After extracting all fluid, is an air space left in tip's end? (The air can be displaced and actual
volume determined simply by rotating the volume adjustment to push the fluid to the very end
of the tip. Then, read the volume directly.)
5. If the measurements were inaccurate, repeat the exercise to obtain a nearly perfect result.
Part 4: Pipette-Mixing Activity
Mixing two liquids together is an essential laboratory skill. Sometimes you turn the task over to a vortexer—a
small vibrating plate. Other times, you need to create a more gentle and controlled mixing motion. Therefore,
you need to be able to mix two solutions with a micropipette. When pipette-mixing, you need to balance mixing
a solution completely with not mixing too vigorously. Over-zealous suction can pull solution into the barrel of
the pipette (cleaning a pipette barrel is an expensive process!) This activity allows you to see and improve your
pipette mixing skills for your upcoming lab activities.
Materials:
1.5 ml microcentrifuge tube
Blue and yellow loading dyes
P-200 and rack of yellow tips
1. Add yellow loading dye. Using a P-200 micropipette, add 100 ul of yellow loading dye into a 1.5 ml
microcentrifuge tube.
2. Add blue loading dye. Add 100 ul of blue loading dye into the same microcentrifuge tube as the yellow
dye. Insert the pipette tip the whole way to the bottom of the tube and expel the blue dye underneath
the yellow dye.
3. Homogenize dyes. In order to make mixing easier, reset your P-200 to 200 ul so that you can suck up
and expel the entire contents of the tube at once. This will also allow you to check your pipetting
accuracy, as you did in the previous activity. Mix the contents of the tube together until completely
homogenized by slowly stirring with the pipette while sucking up and expelling the dyes in the tube.
Once finished, the dye solution should be completely green with no blue or yellow swirls.