1.3: Micropipetting

Learning Objectives

Goals:

• Use various instruments found in the biotechnology lab.
• Measure volume with precision and accuracy.
• Pipet with precision and accuracy.
• Learn how to use a micropipette to measure very small volumes.

Student Learning Outcomes:

Upon completion of this lab, students will be able to:

• Make accurate and precise measurements with micropipettes and serological pipettes.
• Calculate percent error for a given measurement.
• Read, set, and operate a micropipette.
• Determine which pipette should be used to measure a specific volume.
• Determine how accurately you can measure with each micropipette.

Introduction to Micropipetting:

The ability to measure very small amounts, microliters (µl), of liquid chemicals or reagents is a fundamental skill needed in the biotechnology or research lab. Scientists use a device called a micropipette to measure these very small volumes with accuracy. This activity introduces the technique of micropipetting. Remember, as with all fine motor skills, this new skill will require practice and determination. Be sure to operate the micropipette slowly and carefully.

Part I: Choosing and Setting the Micropipette

Figure 1. Labeled parts of a micropipette, front and back.

There are several sizes of micropipettes used in the biotechnology lab. Today, you will be using the P-1000, P-200, and P-20. The P-1000 measures volumes between 100-1000 µl, the P-200 measures volumes between 20-200 µl, and the P-20 measures volumes in the 2-20 µl range. It is important to always pick the correct micropipette for the volume to be measured.

Looking at Figure 3.1, you can see that each micropipette has a similar but different display window. For the P1000, the red number indicates the thousands place, followed by the hundreds, tens, and the ones displayed as small vertical lines. Each line represents 2 µl. The P-200 is reads differently. The display from the top down reads, hundreds, tens, ones, and the vertical lines are considered 0.2 µl. Finally, the P-20 can be read from the top down tens, ones, and the red tenths.

Figure 2. Reading a micropipette at various sizes.

A. Choosing your Micropipette

For each amount listed below, indicate the correct micropipette needed to measure the volume accurately then set the pipette to the indicated amount and show your partner.

Table 1. Pipette Size Choice

Amount	Pipette Needed		Partner Observation
567 µl:
2. 160 µl:
3. 700 µl:
4. 25 µl:
5. 15 µl:

B. Setting your Micropipette

Materials

• P-20 micropipette
• P-20 tips
• Waste container
• Tube of red dye in tube rack
• Laminated sheet for pipetting

Procedure

1. Each student will load 5, 10, 15, and 20 µl of red dye onto the laminated sheet.
2. Locate the p-20 and set the dial to 5 µl.
3. Hold the micropipette in your dominant hand, and gently but securely place the end of the micropipette into the proper size tip. Once the tip is on, be careful not to touch the tip on anything! If your tip touches the bench, lab coat etc. eject the tip into the waste container and get a new clean pipet tip.
4. With your other hand, open the cap of the tube of red dye and bring the tube of red dye to eye level,
5. Push the micropipette plunger down to the first stop and hold your thumb in this position.
6. Place the pipet tip into the red dye solution.
7. Gently release your thumb from the plunger to draw fluid into the tip.
8. Confirm that the tip has liquid and that no bubbles are present within the tip.
9. Close the tube of red dye and place back in tube rack.
10. Gently touch the tip to the center of the circle labeled 5 µL and slowly push all the way down (to second stop) on the plunger to dispense the liquid.
11. Repeat this process for the remaining volumes.
12. Be sure to watch your groupmates to provide feedback and help with their technique.

Results

Take a picture or draw a picture of your spots and include this in your lab notebook as Figure 1. Make sure the figure has a title.

Conclusion

1. Observe if your spots were similar in size to your groupmates.
2. Which volume had the most variability?
3. What could have contributed to your spot being too large or small?

Part II: Pipetting Practice

A. Microplate Art

Materials

• p20 pipette (1)
• p200 micropipette (1)
• P-20/P-200 tips
• Microplate art set (design cards, colored dyes, and 96-well microplate) (1)
• Analytical or electronic balance

Procedure

1. Obtain a 96-well microplate, a design card, and tubes of colored dyes.
2. Write the Microplate Art Design number in your lab notebook.
3. Using the gram balance, obtain the weight of your 96 well microplate and record in your notebook.
4. Using the p200 micropipette with tip, dispense 50 µl of dye into the wells written on the design card.
5. Once you have finished pipetting, weigh your completed microplate, and record in your lab notebook.

Results

1. Be sure to record your weight in grams of your microplate pre/post pipetting in your lab notebook.
2. Using these values, calculate your percent error of the microplate you just created. Include the calculation in your lab notebook.
3. Take a picture of your microplate design and include this in your lab notebook.

CONCLUSION

1. Was your percent error below +/- 5%? If your percent error was above this range, elaborate on the potential causes.
2. Did your pattern look correct? How could you avoid errors in the future?

B. Micropipette Practice Matrix

Materials

• p20 pipette (1)
• p200 micropipette (1)
• 1.5ml microfuge tubes (3)
• Permanent marker
• Analytical or electronic balance

Procedure

1. Label three microfuge tubes: 1, 2, 3,
2. Weigh each tube before placing any liquid inside.
3. Draw table 2 in your lab notebook and use it to record your data.

Results

Table 2. Calculating Accuracy for Micropipetting

Tube #	Weight of tube (g)	Weight of tube + dye (g)	Theoretical weight of dye	Actual weight of dye	% Error
1
2
3

4. Deliver the volumes indicated in Table 3 into each of the 3 labeled tubes.

Table 3. Volumes to be Pipetted into each Tube

Tube #	Micropipette	Red Dye (µl)	Blue Dye (µl)	Green Dye (µl)
1	P1000	210	435	332
2	P200	110	153	67
3	P20	15	17	10

5. Weigh each tube after pipetting.
6. Determine the theoretical weight of the dye using the information about the weight of a mL of the dye solution at room temperature provided by your instructor.
7. Determine the % error for each tube.

Conclusion

Based on your data comment on the following in your lab notebook:

1. Which micropipette gave the most precise measurement?
2. Which micropipette gave the most accurate measurement?
3. What may have contributed to higher percent errors?

Study Questions

1. Convert the following:
   • 345 mL = __________________ µl
   • 0.54 mL = _________________ µl
   • 5.2 L = ________________ mL
2. Which micropipette would you choose to measure 550µl? 17µl? 167µl?
3. Make 3 suggestions that other biotechnologists can use to improve micropipetting accuracy.
4. Assuming that the density of water is 1 gram per milliliter, how much should 550 µL of water weigh?
   • 17 µL of water?
   • 167 µL of water?
5. What is the formula to calculate percent error?
6. What is the maximum volume you can set for each micropipette (P-1000, P-200, P-20)?