Measuring Small Volumes and Mass
EXPERIMENT 1
Materials
Lab coat
Lab notebook
Lab handout
Micropipette, P-10 with tips
Micropipette, P-20 with tips
Micropipette, P-200 with tips
Micropipette, P-1000 with tips
Microcentrifuge
Gloves
dH2O
Eppendorf Tubes
Tube racks
Permanent marker
Balance
Weigh papers
Beaker for used tips
BACKGROUND READING
Very tiny amounts of chemicals and biological reagents are used in many biotechnology
experiments. To measure these minute volumes, technicians use micropipettes that measure
microliter (μL) amounts. Review the following metric conversions:
Liter
Milliliters (ml)…10-3 of a liter---0.001 of a liter!
-6 of a liter---0.000001 of a liter!
This activity introduces micropipetting technique. As with all fine motor skills, learning how to
use a micropipette takes practice and determination. You must be able to measure these very
tiny volumes with accuracy and precision. Operate the micropipette slowly and carefully.
Picking and Setting the Micropipette
1. Check that you have the correct micropipette for the job. Most labs have three sizes of
micropipettes: a P-10 (for 0.5 to 10 μL), a P-100 (for 10 to 100 μL), and a P-1000 (for 100
to 1000 μL).
Note: Some laboratories have a P-20 (for 2 to 20 μL) or a P-200 (for 20 to 200 μL).
2. Dial the desired volume. Understand how to read the scale. Hint: By knowing the
maximum volume of the micropipette, you can figure out what each of the digits on the
readout means.
3. Push the end of the pipette into the proper-size tip. Sometimes the tips or the boxes
they come in are color coded. For example, white tips are for P-10s; yellow are for P20s, P-100s, and P-200s; blue are for P-1000s. Tips are disposable and are intended for
one use only.
Measuring Small Volumes and Mass 1
How to Take Up a Sample with a Micropipette
4. Before picking up the micropipette, open the cap or lid of the tube from which you are
taking fluid. (Skill will eventually allow you to open tubes with one hand.)
5. Hold the micropipette in one hand, at a 45º angle from vertical. In this way,
contaminants from your hands or the micropipette will not fall into the tube. Hold the
container in your other hand. Both should be almost at eye level.
6. Depress the plunger of the micropipette to the first stop, and hold it in this position.
7. Place the tip into the solution to be pipetted.
8. Draw fluid into the tip by slowly releasing the plunger.
How to Expel a Sample from the Micropipette
9. With your other hand, open the cap or lid of the tube you are filling.
10. Hold the micropipette in one hand, at about a 45º angle from vertical. Hold the tube in
your other hand. Both should be about eye level.
11. Gently 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 that helps draw the
fluid out of the tip.
12. Slowly, depress 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 from the tube, keeping the plunger depressed to avoid
drawing any liquid back into the tip.
14. Always change tips for each new reagent you pipette. To eject a tip, depress the ejector
button on the top of the micropipette.
Precautions for Using Micropipettes
 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. Failure to do this will cause liquid to enter
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 leakage or bubbles that will
trap air and make the measurement inaccurate.
Procedures
Part A. Pipetting Demonstration
Part B. Get Ready
1. As specified in the Laboratory Notebook Preparation handout, setup your lab notebook
for Experiment 1.
Measuring Small Volumes and Mass 2
2. Put on a lab coat and gloves and wear them throughout all procedures.
Part C. Practicing with a P-1000
1. Label an empty eppendorf tube as tube A.
2. To tube A, use a P-1000 to add the volumes of water shown in Table 1. Add each
volume to the inside of the tube without letting the drops touch. The drops may stick
because of adhesion. When all volumes have been added, bring the drops to the
bottom of the tube with a quick wrist flick or gentle tapping on the lab bench.
3. Spin tube A in the microcentrifuge to pool any remaining volume in the bottom of the
eppendorf tube. The instructors will show you how to use the microcentrifuge.
4. Check the accuracy of your technique with the P-1000. Set the micropipette to 810 μL
and withdraw the contents of tube A. The contents should just fill the tip, with no air
space at the bottom of the tip, and no leftover fluid in the tube.
5. Record the results in your lab notebook.
Table 1. Volumes of water to add for P-1000 practice.
Reaction
Volume I
Volume II
Tube
A
253 μL
557 μL
Part D. Practicing with a P-200
1. Label an empty eppendorf tube as tube B.
2. To tube B, use a P-200 to add the volumes of water shown in Table 2. Add each volume
to the inside of the tube without letting the drops touch. The drops may stick because
of adhesion. When all volumes have been added, bring the drops to the bottom of the
tube with a quick wrist flick or gentle tapping on the lab bench.
3. Spin tube B in the microcentrifuge to pool any remaining volume in the bottom of the
eppendorf tube.
4. Check the accuracy of your technique with the P-200. Set the micropipette to 98.3 μL
and withdraw the contents of tube B. The contents should just fill the tip, with no air
space at the bottom of the tip, and no leftover fluid in the tube.
5. Record the results in your lab notebook.
Table 2. Volumes of water to add for P-200 practice.
Reaction
Volume I
Volume II
Tube
B
22.3 μL
31.6 μL
Volume III
44.4 μL
Part E. Practicing with a P-20 and P-10
1. Label an empty eppendorf tube as tube C.
2. To tube C, use a P-20 to add the volume I of water shown in Table 2. To tube C, use a P10 to add volumes II and III of water shown in Table 3. Add each volume to the inside of
the tube without letting the drops touch. The drops may stick because of adhesion.
Measuring Small Volumes and Mass 3
When all volumes have been added, bring the drops to the bottom of the tube with a
quick wrist flick or gentle tapping on the lab bench.
3. Spin tube C in the microcentrifuge to pool any remaining volume in the bottom of the
eppendorf tube.
4. Check the accuracy of your technique with the P-20. Set the micropipette to 12.0 μL
and withdraw the contents of tube C. The contents should just fill the tip, with no air
space at the bottom of the tip, and no leftover fluid in the tube.
5. Record your results in your lab notebook.
Table 3. Volumes of water to add for P-20 and P-10 practice.
Reaction
Volume I
Volume II
Tube
C
6.5 μL
Volume III
4.3 μL
2.2 μL
Part F. Checking the Accuracy of Your Micropipetting Using a Balance
A balance can be used to determine if a micropipette is measuring within an acceptable range.
Since 1 mL of water weighs 1.0 g, you can estimate the expected mass for any volume of water.
For example:
1.5 mL of water should weigh 1.5 g.
0.25 mL of water should weigh 0.25 g.
150 μL (= 0.15 mL) of water should weigh 0.15 g.
Water dispensed by a micropipette can be weighed on a balance. By comparing the actual
observed mass to the expected mass, you can make an error determination. For any
measurement, calculate the % error using the following equation:
(observed mass – expected mass) x 100 = % error
expected mass
1. For each micropipette, measure the specified volume of dH2O onto a piece of weigh
paper on a “tared” balance. The instructors will show you how to use the balance.
Make a copy of Table 3 below for your notebooks, and in Table 3 record the mass of the
volume of water measured and calculate the % error.
Table 3. Pipetting precision: mass versus volume.
Micropipe
Volume
Volume
Expected Observed
tte
(μL)
(mL)
Mass (g)
Mass (g)
% Error
Acceptable
Error (%)
P-1000
257.0
3
P-200
53.0
5
P-20
20.0
5
Measuring Small Volumes and Mass 4
Part F. Finishing Up
1. Take a look at your pipetting results today, and in your lab notebook, record any final
thoughts you have on your micropipetting technique.
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