Adapted from 1994 Gene Connection Version 1.5 –
Techniques Lab A: Manipulating Small Volumes
Micropipette Techniques Lab: Manipulating Small Volumes
Background
A forensic scientist extracts a miniscule 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 chemist, the genetic engineer, and the criminologist perform laboratory
procedures involving tiny amounts of DNA and chemicals, each utilizes 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. Recall that
‘micro-‘ is a prefix in the metric system which means “one-millionth” of the base unit (in this case, a
liter, “L” or “l”). It may be easier for you to picture one milliliter (mL or ml) of water. If you mentally
subdivide that milliliter of water into 1000 tiny equal-sized volumes, each volume is one microliter
(abbreviated µL or µl).
Purpose
The laboratory activity introduces micropipetting technique. As with all fine motor skills, learning how
to use a micropipette takes practice and determination. You will be awarded with excellent DNA
laboratory results.
Materials per team of 2
Solution 1, colored
Solution 2, colored
Solution 3, colored
Solution 4, colored
Solution 5, clear
GG solution, glycerol
P-20 micropipette and yellow tips
P-200 micropipette (optional)
supply of 1.5 mL reaction/Eppendorf tubes
rack for reaction tubes
container for waste (tips)
paper towels
fine- tip permanent marking pen
transparency
microcentrifuge (also called microfuge)
Directions for using Micropipettes
Turn your micropipette to 20 µl. Depress the stopper to the 1st stop and “feel” the distance. Now turn
your micropipette to 2 µl. How does the distance to the 1st stop compare to the distance at 20 µl?
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 because you won’t draw
accurate volumes.
 When using a micropipette, first apply a tip. Forgetting to do this would cause liquid
leakage 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 a fluid in the tip. Do not
allow liquid to accidentally run back into the nose cone.
 Using your thumb to control the speed at which the plunger rises after taking up or
ejecting fluid. Releasing the plunger to abruptly will cause liquid to pop up into the nose cone.
Part I Procedure:
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 20-200µL), and a “P-1000” (for 200-1000 µL).
2. Dial the desired volume. Do you understand how to read the scale? If not- ASK!
3. Push the end of the pipette into the proper size tip. The yellow tips are for P-20’s and P 200’s;
the large blue tips are for P-1000’s.
How to Take Up Sample with a Micropipette
4. Before picking up the micropipette, open up the cap or lid of the tube from which you are
taking fluid. (Or have your lab partner do this)
5. Hold the micropipette in one hand, almost vertical; hold the tube in your other hand. Both
should be almost eye level.
6. Depress the plunger of the micropipette to the first stop, and hold it in this position then...
7. Dip the tip into the solution being pipetted. Make sure you are holding this eye level.
8. Draw the fluid into the tip by slowly releasing the plunger.
Depress (put it in the reaction tube) then release (then pull it out of the reaction tube).
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 reaction 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 eye level.
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 depress the plunger of the micropipette to the 1st 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 depressed to avoid sucking any
liquid back into the tip.
(In the tube) Depress, depress (remove), release.
14. Always change tips for each new reagent you need to pipette. To eject a tip, depress the large
button on the top-side of the micropipette.
Activity: Practice with the Micropipette
Practicing with the P-20 – be sure you have a tip on your micropipettor!
1. Practice by following the directions above by withdrawing 2 µL from one of the solution tubes
(1-5) and expelling it onto your transparency. You and your partner’s drop should be identical
in size.
2. Again practice, this time withdrawing and expelling 20 µL. Be sure to change the tip if you are
using a different solution.
3. Label two empty reaction tubes “A” and “B” with the permanent marker.
4. Add the amounts of solutions 1, 2, 3, and 5 into tubes A and B as shown in the table below. It
is probably easiest for one person to dispense Solution 1 in both A and B tubes and your partner
to dispense Solution 2 into both A and B tubes, and so on.
5. When you are adding several reagents to one tube, release each drop onto a new location on the
inside wall of the reaction tube. This will help you keep track of what you have added.
Reaction Tube
A
B
1
4 µL
6.5 µL
2
5 µL
2.5 µL
3
2 µL
---
4
-----
5
--2 µL
Check with Ms. Bolton – you may be skipping this.
6. Spin tubes A and B in the microcentrifuge for 3 seconds to pool the solutions. REFER TO
CENTRIFUGE INSTRCUTIONS AT THE BOTTOM OF THIS PAGE.
7. Set the micropipette to 11 µL. As a check of your technique, withdraw all of the fluid in tube A.
The contents should just fill the tip – no air space at the bottom of the tip and no leftover fluid
in the tube. If you have bubbles or spaces within the fluid in the tip, you need to withdraw the
fluid more slowly and make sure you did not move the tip away from the liquid source.
8. Expel the fluid onto the transparency.
9. How much should be in tube B? Check your technique by setting the pipettor to the correct
volume and withdraw all (hopefully) the solution in tube B.
10. Expel the fluid onto the transparency next to the fluid from tube A. These drops should be
identical in size.
Practicing with the P-200 – be sure you have a tip on your micropipettor!
1. Label an empty reaction tube “C” with the permanent marker.
2. Add the amounts of solutions 1, 2, and 4 into tube C as shown in the table below.
Reaction Tube
C
1
20 µL
2
40 µL
3
---
4
100 µL
5
---
3. Spin tube C for 3 seconds. You will need to spin your tube with another group's to balance the
centrifuge.
4. Check the accuracy of your micropipetting technique with the P-200. Set it to _____ µL (total
volume) and withdraw the content from tube C. Does it just fill the tip? No air bubbles?
5. Expel the volume onto the transparency.
Centrifuge Instructions:
1. Tightly close the caps on all of the tubes to be placed in the microcentrifuge (also called
microfuge).
2. The microfuge rotor must always be balanced – you cannot, for example, insert one tube into
the machine. Spinning an unbalanced arrangement like this would damage the motor.
3. The amount of liquid in the tubes should be similar, otherwise the rotor will spin unevenly (like
wet towels 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:
Part II:
Background:
4. After you have closed the lid of the microfuge, give the tubes a 3 second pulse. This will mix
and pool all the reagents into a droplet at the bottom of each tube.
Part II: Practice with Viscous Solutions
Enzyme and DNA Solutions are often viscous due to high concentrations of protein, DNA, or a
stabilizing agent such as glycerol. Viscous solutions require extra care in pipetting in order to deliver
accurate amounts of sample and not to waste precious materials.
Purpose: to refine your pipetting technique, working with tiny volumes and viscous solutions.
REVIEW:
 Set pipette volume only within the range specified for the micropipette.
 When using a micropipette, first apply a tip.
 Always keep the micropipette in a vertical position when there is fluid in the tip.
 Use your thumb to control the speed at which the plunger rises after taking up or ejecting the
fluid.
TO EMPHASIZE:
 To fill:
Depress (put it in the reaction tube) then release (then pull it out of the reaction tube).

To expel:
(In the tube) Depress, depress (remove), release.
Activity: Practicing with Viscous Solutions
1. Check that you have the P-20 micropipette. Place a tip on the end so you don’t forget to do so
later.
For step 2 you are going to use the pipette INCORRECTLY – THIS TIME ONLY!
2. Dial 2 µL. Push the plunger to the SECOND stop and withdraw a sample of the glycerol (GG)
solution.
3. Look at the volume in the tip and draw a picture of the liquid level in the tip on your answer
sheet.
4. Deliver this incorrect volume to a spot on the transparency. Draw the actual size of the drop
next to your tip drawing.
For the rest of the assignment, use the pipette CORRECTLY!
5. With the P-20 set to 2 µL, push the plunger to the 1st stop and withdraw a sample of the GG
solution.
6. Look at the volume in the sample tip and draw an accurate picture of the liquid level on your
answer sheet.
7. Deliver this correct volume to one spot on the transparency and draw the actual size of the
drop.
8. If this second drop is actually 2 µL, estimate how many µL are in the 1st “incorrect” drop.
Write this on your answer sheet.
Determining Volume of Sample in “Incorrect” Drop
9. With the P-20 still at 2 µL, correctly remove 2 µL, from the “incorrect” drop and place it
elsewhere on the transparency.
10. Remove a second and third 2 µL from the original “incorrect” drop and place them along side
the first. Some of you may even be able to get a 4th 2 µL from the drop.
11. Write your more accurate estimate of the “incorrect” drop on your answer sheet.
12. If 2 µL represents a 100% sample, what % was represented by your incorrect drop?
If there is extra time
13. Use the pipette to correctly sample 4, 6, and 10 µL of the glycerol.
14. Draw the three pipette tips and indicate the volume of each on the last blank spot on your
answer sheet in addition to the size of the drop on the transparency.
15. If Ms. Bolton asked you to look at a pipette tip with a small sample in it, would you be able to
guess the amount in µL? What about a drop on paper? What about in a tube?
16. Look at the sample in the front of the classroom. What volume do you estimate it contains?
Upon completion of this lab:
 Dispose of all used tips and reaction tubes in the waste container.
 Use a paper towel to wipe off and dry the transparency sheet.
 Replace the used reaction tubes with fresh ones.
 Leave all equipment as you found it.
 Finish your lab sheet.
Micropipette Techniques Lab
Pre-lab:
1 L = ________ ml
Micropipettor Range
Micropipettor Max
Setting
Amount in L
Amount in mL
Amount in µL
1 ml = _______ µL
Name: ______________________
Per: ____ Date: _______
1 µL = ________ L
2-20 µL
20-200 µL
200-1000 µL
20 µL
200 µL
1000 µL
Part II – Practice with Viscous Solutions:
#3
#4 Drop
#7 Drop
#6
#8 Estimate: _______
#11 New estimate: ______
#12 _______%
Write down how many µL you think are in the sample Bolton has at the front of the room. _____
More Lab Practice:
4. Select the appropriate micropipette and show what the dial should read to measure each of the
following amounts of liquid. Write the amount on the line beneath each drawing.
5. Why is it important to balance the centrifuge before turning the motor on?
_______________________________________________________________________________
6. Show how you would arrange the given number of tubes in each centrifuge to balance the load
by coloring in the location they would be placed. I have shaded the 1st for you. You must
decide if you need to add or remove extra tubes, so indicate this on the drawing below.