MLAB 2337 Molecular Diagnostics Techniques
Laboratory 3: Micropipettes
Points: 20
Objectives
1. Identify the parts of a micropipette and state the function of each part.
2. Demonstrate the proper use of micropipettes by:
a. Selecting proper pipette for task
b. Setting the correct volume
c. Proper operation of plunger
d. Selecting proper tip for pipette
e. Exam pipette tip for flaws
f. Attaching and discarding pipette tips
g. Drawing up sample using proper orientation.
h. Dispensing sample
3. State the reason the pipette tip should be immersed only a few milliters below the fluid surface.
4. State what may occur if the plunger is allowed to snap up.
5. State when the following types of tips should be used: sterile, barrier.
6. Demonstrate the proper filling and dispensing of fluids using a micropipette.
7. State 8 precautions which must be followed when using micropipettes.
8. Given a diagram of a micropipette volume readout, correctly state the volume indicated.
Discussion
Micropipettes are used extensively in molecular diagnostics to accurately and precisely deliver small
volumes of liquid. The correct use of micropipettes is essential to ensure accurate volume delivery and to
prevent damage to these expensive pieces of equipment. It is crucial that, prior to use, the student become
familiar with function of the parts, function and use of the micropipette.
Most micropipettes have the following basic parts:
1. Volume adjustment knob – turned to adjust volume
2. Volume indicator
3. Plunger button – aspirate and dispense
4. Tip ejector button – discard tip after use
5. Volume adjustment knob
6. Plastic shaft 7. Tip ejector arm – discard tip after use
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Test your knowledge: http://www.public.coe.edu/ibench/pipet/partsquiz.htm . Does not work with FireFox
Adjusting the Volume
Volumes needed for molecular procedures are tiny and proper measuring is essential to ensure accuracy of
results and prevent contamination.
The measuring ranges for the micropipettes most frequently used are:
 P10 - 1.0-10.0µL
 P20 - 2.0-20.0 µL – yellow tips
 P100 -10.0-100.0 µL – yellow tips
 P200 - 20.0-200.0 µL
 P1000 - 100.0-1000.0 µL – should only be used for volumes 200 or greater
The black volume adjustment dial near the top of the micropipette allows you to adjust the volume that is
measured. It can be dialed to the left or right to increase or decrease the volume.
The digital readout shows the volume that will be measured. As you turn the volume adjustment dial, the
numbers in the digital readout will change. The digital readout has three numbers which correspond to
different volumes on the different size pipettes.
 In a P10, the top number refers to 10’s of µL, the middle number refers to µL, and the bottom
number refers to 1/10th of µL. Numbers are typically black-black-red. The change in color indicates
the position of the decimal point.
 In a P20, the top number refers to 10’s of µL, the middle number refers to µL, and the bottom
number refers to 1/10th of µL. Numbers are typically black-black-red. The change in color indicates
the position of the decimal point.
 In a P100 the top number refers to 1000’s of µL, the middle number refers to 100’s of µL and the
bottom number refers to 10’s of µL. Numbers are all black.
 In a P200, the top number refers to 100’s of µL, the middle number refers to 10’s of µL and the
bottom number refers to µL. Numbers are all black.
 In a P1000, the top number refers to milliliters. Numbers are typically red-black-black. Note that
this pipette reads milliliters while the others read microliters.
When looking at the volume display the black digits indicate microliters; the red digits indicate tenths and
hundredths of microliters. On P1000 micropipettes, the red digits indicate milliliters and the black digits
indicate microliters.
Never adjust a micropipette outside of the range!
.When setting the volume always dial down to the volume setting. This prevents mechanical back lash
from affecting accuracy. Otherwise precision and accuracy will be affected.
Pipette Tips
Liquids are never drawn directly into the shaft of the pipette. Instead, disposable plastic tips are attached to
the shaft. There are different types of tips. Select the appropriate tip for the pipette used.
There are several different types of tips available. Barrier tips provide the best protection from air
contamination, but they do not always fit on the smallest micropipettes. Sterile tips are used to ensure that
samples are not contaminated. Do not use sterile tips for non-sterile applications. Gel loading tips are used
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to load samples into gel for electrophoresis. Do not use gel loading tips for non-gel-loading applications.
These tips are expensive.
The tips are racked in plastic boxes with covers. Be careful to not touch the tips to prevent contamination.
The box should be closed when not in use to prevent airborne contamination.
Tips must seal properly on the shaft to assure an airtight seal and avoid leaks or poor accuracy.
Tip - General
1. Tips must seal properly on the shaft to assure an airtight seal and avoid leaks or poor accuracy.
2. Tips must be soft and flexible so that the shaft is not scratched or worn prematurely.
3. Tips must be free from microscopic particles.
4. The tip orifice must be the correct size, and orifice size and geometry must be consistent from tip to tip.
5. Interior and exterior surfaces must be clear, smooth and hydrophobic to avoid retention of liquid.
Inserting tip:
1. Select the correct size tips.
2. Open box without touching the tips with your hands.
3. Insert the micropipette shaft into the tip and press down firmly. This will attach the tip to the shaft.
4. Close the box without touching the tips with your hands.
To avoid contamination a fresh tip should be used for each measurement. Never immerse the tip more than
a few millimeters below the surface of the fluid. A deeper immersion can cause fluid to cling to outside of
tip, delivering more fluid than was desired.
Tips should be discarded in a cardboard biohazardous waste container.
Plunger Settings
There are two stops on a micropipette plunger:
1. The plunger is slowly depressed to the first stop to aspirate the sample. This pushes air out of the
pipette tip in preparation for aspiration of the correct volume of fluid. This is where initial
resistance. The tip is inserted into the liquid and the plunger slowly released to aspirate the sample.
2. The second stop is to dispense, the plunger is carefully pushed down beyond the initial resistance
until it is in contact with the body of the pipette. Applying pressure to the second stop results in
dispensing the correct volume of fluid.
Once the sample is dispensed depress the tip release button located near the plunger to discard the pipette
tip.
View the video on correct use:
 http://www.youtube.com/watch?v=8Afh_0IAfrQ&feature=related
 http://www.youtube.com/watch?v=waZfBR7fk_8&feature=related
The plunger should be depressed in a smooth motion to the first stop. This is the position of initial
resistance and is the level of depression that will result in the desired volume of liquid being transferred.
Never let the plunger snap up. This can cause air bubbles to fill the tip, destroying accuracy and may
eventually lead to damage to the micropipette.
Always keep the micropipette in vertical position to ensure that no liquids enter the micropipette or drip
from the disposable tip. Never the lay the micropipette down when liquid is in the tip. This may allow
Laboratory 3 Micropipettes
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fluid to enter the pipette and ruin it. Dispense the fluid into the lowest possible place in the receiving
vessel.
Use of Micropipette
1. Depress plunger to first stop.
2. Immerse tip approximately 3mm into the sample solution (Step 1).
3. SLOWLY release the plunger until it reaches the original position (Step 2). Watch as the solution is
drawn up into the tip. DO NOT release plunger quickly as this may result in air bubbles being
drawn up or splashing solution on the non-sterile shaft.
4. Withdraw tip from solution and place tip against the side wall of receiving container (Step 3).
5. Smoothly depress the plunger to the first stop (Step 4) and continue to depress until the second stop
is reached (Step 5).
6. Remove the tip from the receiving container and slowly allow release the plunger. DO NOT let the
plunger snap back into place.
7. Remove the disposable tip by depressing the tip ejector button (Step 6).
Precautions
Micropipettes are expensive! To keep them functioning properly it is crucial that they be handled with
care. Please follow these rules to keep them in working order:
1. Never adjust the volume beyond the range of the micropipette. No micropipette should be adjusted
below zero µL. The P20 should never be adjusted above 20 µL , the P200 over 200 µL and the
P1000 over 1 mL
2. Never force the volume adjustor dial. If the knob becomes difficult to adjust it probably means that
you are exceeding the limits for the pipette or the pipette is damaged. Report the problem to your
instructor.
3. Do not drop the micropipette.
4. Always use a smooth motion when using the micropipette. This will help give accurate
measurements and also prevent breakage of the pipette. There should not be “snapping” noises.
5. Always keep pipettes upright.
6. Never lay a pipette with liquid in the tip on the bench.
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7. Always choose the appropriate size pipette for the volume you are measuring.
8. Always dispose of tips in the appropriate waste receptacle.
Validation
Micropipettes must be validated on a regular basis to ensure delivery accuracy. There are several different
methods with gravimetric and colorimetric (spectrophotometric) being the most common.
In the gravimetric method a balance is used, preferably one with a draft shield since the volumes are so
small. The micropipette is set at a volume and this volume is dispensed 10 times into a weighing pan
which has been tared. Each time a volume is dispensed the weight is recorded.
In the colorimetric method a blank and dye solution are used. A colored solution is pipetted into a volume
of water, followed by measuring the absorbance of the solution.
Micropipettes
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USE OF VWR MICROPIPETTES P10, P20, P100, P200 AND P1000
Updated May 19, 2006
Appearance of Analog Readout at Minimum and Maximum Volumes of Micropipettes
P1000 (100-1000 μL)
Minimum, 100 μL
0
1
0
Maximum, 1000 μL
0
2
0
Maximum, 1000 μL
0
2
0
Maximum, 200 μL
2
0
0
0
5
0
Maximum, 200 μL
2
0
0
1
0
0
P1000 (200-1000 μL)
Minimum, 200 μL
1
0
0
P200 (20-200 μL)
Minimum, 20 μL
P200 (50-200 μL)
Minimum, 50 μL
P20 (2-20 μL)
Minimum, 2 μL
0
2
0
Maximum, 20 μL
0
1
0
Maximum, 10 μL
2
0
0
P10 (1-10 μL)
Minimum, 1 μL
Laboratory 3 Micropipettes
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0
0
6
PROCEDURE
Supplies
1. Set of micropipettes
2. Pipette tips appropriate for micropipette
3. microtubes
4. colored solution
Procedure
1. Practice with a P100, P20 and P10
2. Select a micropipette from your set that will measure the volume you need.
3. Set volume using volume adjustment knob. Hold micropipette in one hand, and with other hand, turn
volume adjustment knob counterclockwise so volume indicator is 1/3 revolution above desired setting,
then turn slowly clockwise until indicator shows desired volume. If micropipette dial is past either its
high or low limits, or the dial will not rotate, notify your instructor immediately.
4. Practice adjusting the volumes on each of the micropipettes (P100, P20 and P10). Look carefully at the
numbers and review the chart to determine what volume the micropipette is set at.
5. If you pass the desired setting, turn dial 1/3 revolution higher than desired and reset volume.
6. Attach a new disposable tip to the pipette shaft by pressing shaft into tip while tip is still in box. Press
only hard enough to make a positive airtight seal. Close cover on box of tips.
7. Practice finding the two stops on the micropipette by slowly depressing the plunger.
8. Practice drawing and releasing fluid. Press plunger to first stop. This part of the stroke is volume
indicator.
9. Holding micropipette vertically (perpendicular to the bench), immerse the last few millimeters of the
disposable tip in the colored fluid to be measured.
10. Release the plunger slowly until it has returned to its original position.
11. Pause for a few seconds to ensure that the full volume of fluid is drawn into the tip.
12. Withdraw the tip from the sample, keeping the micropipette completely vertical.
13. Inspect tip to make sure there is no air bubble on the inside and there is no solution on the outside of the
tip.
14. If there is an air bubble inside tip, you must pipette this volume back into the sample and measure
again, repeating steps 9-14.
15. If there is solution on the outside of the tip, touch tip to the wall of the solution container to remove it.
16. To dispense the sample touch the tip end against the side wall of the receiving vessel and depress the
plunger slowly to the first stop, then press the plunger to the second stop, expelling any residual liquid
in the tip.
17. With the plunger fully pressed, withdraw pipette from the vessel carefully, with the tip remaining
against the wall of the vessel.
18. Release the plunger to return to the up position.
19. Use proper technique to draw and dispense the colored solution with each of the micropipettes three to
five times each, resetting the volume control each time.
20. Discard the tip into a waste beaker or trash receptacle by depressing the tip ejector button.
21. Demonstrate to your instructor the dispensing of a given volume of solution.
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Name ________________________________________
Date ___________________
MLAB 2337 Molecular Diagnostics Techniques
Laboratory 3 Micropipettes
Laboratory Results
Points:
/20
Instructions:
1. The student will practice the skill 3-5 times prior to being evaluated.
2. The instructor will state the volume to be dispensed by the student.
3. The student will select the pipette which will most accurately deliver the volume to be dispensed.
4. The student, under direct observation, will draw up and dispense the volume requested.
5. The instructor will place a check mark in the appropriate column.
6. If a “NO” is checked for any skill the instructor will remediate the student and allow an additional
attempt.
Volume to be dispensed: _____________
Pipette selected: _________________
1.
2.
3.
4.
5.
6.
Skill
Correct micropipette used.
Micropipette set to correct volume.
Tip applied securely to micropipette.
Closed tip box.
Correct technique used for aspirating and dispensing.
Ejected tip properly into approved container.
__________________________________________
Instructor Signature
Laboratory 3 Micropipettes
YES
NO
___________________
Date
8
Name ________________________________________
Date ___________________
MLAB 2337 Molecular Diagnostics Techniques
Study Questions
Laboratory 3 Micropipettes
Points:
/22
Instructions:
 Copy/paste the following study questions into word processing document.
 Answer the questions
 Save the file as “Lab4MicropipettesYOUR NAME”
 Submit to “Assignments” in BlackBoard
Study Questions
Write the correct term used for each of the numbered items on this micropipette. (3.5 points)
1.
2.
3.
4.
5.
6.
7.
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8. State the measuring range for each of the following micropipettes (4 points):
a. P10
b. P20
c. P100
d. P200
9. After attaching a pipette tip to the micropipette what should be done to the box of tips and WHY this
must be done? (1 point)
10. Describe the purpose of the two stops on a micropipette plunger. (2 points)
a. First stop
b. Second stop
11. Explain why the micropipette plunger should never be allowed to snap back in place. (1 point)
12. Why should the pipette tip never be immersed more than a few millimeters below the surface of the
fluid? (1 point)
13. How is the tip removed from the micropipette? (1 point)
14. State the volume, using the correct units, of the following readout on a P10 micropipette (1 point):
0
2
0
15. List 8 precautions which must be followed when using a micropipette. (8 points)
a.
b.
c.
d.
e.
f.
g.
h.
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