8: Micropipetting

Pre-Lab: Micropipetting Mastery

Objective:

Before starting the lab, students will understand the purpose of micropipetting, its applications, and the principles of accurate liquid measurement. This section prepares students for precise pipetting techniques and error analysis.

Background Knowledge:

• Why Micropipettes Matter:
  • Micropipettes allow accurate and precise measurement of microliter volumes in laboratory experiments.
  • They are commonly used in molecular biology, enzymatic assays, and cell culture work.
• Types of Micropipettes:
  • P-1000: Measures 100-1000 µL (used for buffers and media)
  • P-200: Measures 20-200 µL (used for PCR and DNA/RNA quantification)
  • P-20: Measures 2-20 µL (used for precise enzyme or reagent additions)
• Reading the Volume Display:
  • Understand how to read the digital display for different micropipette models.
  • Identify correct settings to ensure accurate volume transfer.
• Correct Pipetting Technique:
  • Press plunger to the first stop before submerging the tip in the liquid.
  • Slowly release the plunger to aspirate the liquid while avoiding bubbles.
  • Dispense the liquid smoothly against the side of the receiving vessel.
• Common Errors to Avoid:
  • Using the wrong micropipette for a specific volume.
  • Pipetting too fast, causing air bubbles.
  • Not changing tips between different samples (risk of contamination).
  • Mishandling or dropping the micropipette.

Pre-Lab Questions:

• Why is accuracy important when using micropipettes?
• Which micropipette would you use to measure 150 µL? 15 µL? 750 µL?
• Describe the difference between the first and second stop of the micropipette plunger.
• What are some potential errors that could affect pipetting accuracy? How can you prevent them?
• Why is it important to record percent error during pipetting verification?

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Chapter Eight

Micropipetting

Accurately getting the measurements is super important, especially when we're dealing with really tiny amounts of liquids. Micropipettes are important in the lab because they help us do this job with extreme accuracy. When we need to measure tiny volumes of liquids, usually in the microliter range (that's like really, really small amounts), micropipettes are our go-to tools. Unlike regular pipettes where you have to withdraw and dispense liquids manually, micropipettes are way better. They use mechanisms to withdraw and dispense out liquids precisely, leaving very little room for error. Let's learn all about micropipettes - how they work and the best ways to use them in our experiments.

Operating a micropipette may appear simple at first glance, but truly mastering it requires careful attention to detail and plenty of practice. Micropipettes are available in various sizes to accommodate different volume ranges, commonly known as P-1000, P-200, and P-20, which are widely used in laboratories. Each micropipette is designed for specific volume ranges, and it's crucial to choose the appropriate one based on the volume of liquid you need to measure to ensure accurate results. For instance, in a molecular biology lab, you might utilize a P-1000 micropipette for transferring larger volumes of buffer or media, a P-200 for PCR reactions, and a P-20 for precisely adding enzyme or primer solutions. So, when selecting a micropipette, consider the volume you're working with. The P-200 micropipette is typically used for volumes ranging from 20 µl to 200 µl. This range makes it suitable for various applications, such as dilutions, sample transfers, and DNA/RNA quantification. On the other hand, the P-20 micropipette is designed for even smaller volumes, typically ranging from 2 µl to 20 µl. This micropipette is ideal for tasks requiring extreme precision, such as PCR setup, restriction enzyme digestion, and pipetting small aliquots of expensive reagents. So, when selecting a micropipette, consider the volume you're working with and choose the appropriate one to ensure accurate measurements. If you need to transfer 500 µl of a sample, opt for the P-1000 micropipette. This micropipette is capable of measuring volumes between 100 µl and 1000 µl, providing the accuracy and precision needed for your measurements.

Micropipettes also come equipped with display windows that show the volume being measured, and knowing how to interpret these displays is key for accurate pipetting. See Figure 8.1. Understanding the calibration markings on the micropipette is crucial for precise pipetting. These markings indicate different volume increments, so becoming familiar with them is essential.

For instance, let's take a look at the display window of a P-1000 micropipette. You'll notice red numbers indicating thousands, followed by smaller lines representing hundreds, tens, and maybe ones. Similarly, the P-200 and P-20 micropipettes have their own display formats tailored to their specific volume ranges.

Let's explore the withdrawal and dispensing technique. When withdrawing liquid, it's vital to press the plunger down to the first stop. This action creates a vacuum before you put the tip into the liquid. Doing this ensures that you aspirate the desired volume accurately without introducing any pesky air bubbles. It's important to take it slow when you're drawing up the liquid to prevent those air bubbles from forming, as they can mess with the accuracy of your measurements. Now, when it comes to dispensing the liquid, you'll want to press the plunger down to the first stop, just like before. Then, you'll smoothly and steadily release it. To minimize any liquid sticking to the tip, it's a good idea to touch the side of the vessel as you dispense. Another handy technique to remember is airdropping. This involves expelling a tiny bit of liquid from the tip after you've dispensed the main volume. Airdropping helps prevent any contamination and ensures that you deliver the volume precisely where you need it. It's crucial to watch out for air bubbles in the tip because they can throw off your volume measurements. If you spot any bubbles, you'll need to expel the liquid and start the pipetting process again.

Let's explore some real-world applications. In enzymatic assays, getting the volumes of substrates and enzymes just right is crucial for figuring out reaction kinetics and enzyme activity. Accurate micropipetting ensures that the right amounts of reactants are mixed, leading to reliable assay results. For example, when we're studying an enzyme involved in DNA replication, making sure we measure enzyme and substrate volumes precisely helps us determine enzyme kinetics and how efficiently DNA gets copied. PCR is a super common technique in molecular biology, and it relies heavily on precise pipetting of things like DNA templates, primers, and building blocks called dNTPs. If we mess up the pipetting, it can mess up the whole reaction, leading to wonky results. For instance, when we're amplifying a specific DNA sequence for genetic analysis, making sure we measure PCR reagents accurately ensures that we get a reliable and consistent amplification of our target DNA. In cell culture experiments, we need to prepare growth media and reagents with extreme care because accurate pipetting is crucial for keeping our cells happy and healthy. If we're not careful with our pipetting, we can introduce variability in the conditions our cells are growing in, which can mess with our results. For example, when we're growing mammalian cells for drug screening assays, making sure we measure media components precisely ensures that our cells are always in the best conditions for our experiments.

In conclusion, mastering the art of micropipetting is absolutely essential for success in the laboratory. By understanding how micropipettes work, following best practices, and practicing our skills, scientists can ensure that their experiments are precise, accurate, and reproducible. Whether we're doing enzymatic assays, PCR reactions, or cell culture experiments, being really good at micropipetting is key to getting reliable and meaningful results in scientific research.

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LAB ACTIVITY - PART 1 - Verification of Micropipettes

Objective: To verify the accuracy of micropipettes using an electronic balance.

Notes: Keep accurate records of the % error for each micropipette, as this information will be crucial for selecting the appropriate micropipette during a skill evaluation quizzes and practicum.

Materials:

• Micropipettes (P-1000, P-200, P-20)
• Tips compatible with each micropipette
• Distilled water
• Electronic balance
• Weighing boats or beakers
• Laboratory notebook and pen

Procedures:

1. Make sure your micropipettes are clean and ready to use.
2. Have your electronic balance set up and turned on.
3. Tare a measuring cup.
4. Measure with P-1000:
   • Use the P-1000 micropipette to measure water three times for each volume: 1000 µL, 500 µL, and 100 µL.
   • Write down the mass of each measurement from the electronic balance.
   • Calculate the average % error for each volume using the formula: % Error = [(Measured Mass - Theoretical Mass) / Theoretical Mass] x 100.
5. Measure with P-200:
   • Measure water five times for each volume: 200 µL, 100 µL, and 20 µL.
6. Measure with P-20:
   • Measure water fifty times for each volume: 20 µL, 10 µL, and 2 µL.
7. Grab tape, stick it onto the micropipette, and then write down the percent error on the tape. Include the date and your initial
8. Continue with other micropipettes, if any
9. Dispose of any used tips and water properly.
10. Wipe down your micropipettes and electronic balance to keep them clean.

Figure 8.2: Examples of how your data should be collected

Fill out the tables below: 

p1000 Micropipette: 

p200 Micropipette:

p20 Micropipette:

Summary to show in the PowerPoint Slide

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LAB ACTIVITY - PART 2 - Skill Evaluation 1 (20pts)

During this skill evaluation, it's crucial to stress the significance of efficiency and time management. In laboratory settings, time is valuable, and being able to complete tasks quickly without compromising accuracy is essential for productivity.

Objective: To assess students' proficiency in accurately measuring volumes and transferring liquids into conical tubes and microcentrifuge tubes within a specified time frame.

Materials:

• Micropipettes and tips
• Serological pipettes and tips
• Mass standard
• Conical tubes
• Microcentrifuge tubes
• Liquid samples (red, blue, yellow, green)
• Timer
• Grading rubric

Procedure

1. Begin by using the mass standard to determine the absolute error for the scale being used. Calculate the absolute error: _______ g
2. Set up a workstation with all necessary materials arranged neatly and within reach.
3. Preweigh a Conical tube and a Microcentrifuge tube
4. Add the specified volumes of each liquid to the corresponding tubes:
   1. Conical tubes: 6 mL red, 6 mL blue, 8 mL yellow, 30 mL green
   2. Microcentrifuge tubes: 600 µL red, 300 µL blue, 80 µL yellow, 20 µL green
5. Accurately complete the task within the designated time limit of 3 minutes.

For TA or Instructor only:

6. Afterward, post-weigh both the Conical tube and the Microcentrifuge tube.
7. Measure the weight of the final solutions and calculate the percent error for both solutions in the Conical tube and the Microcentrifuge tube.
8. Accumulate the average percent error from both solutions, which will be subtracted from their score of 20 points.
   1. For instance, if their percent error is 8% from the Conical tube and 2% from the Microcentrifuge tube, the average percent error (used for grading purposes) will be 5%, resulting in a total of 15 out of 20 points.

Extra Credits

• Award 2 extra credit points to any student who achieves a percent error of less than 1% on either the Conical tube and/or Microcentrifuge tube. Students may receive up to 4 extra points if they obtain under 1% error for both tests.

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Summary to show in the PowerPoint Slide

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Post-Lab: Reflection and Analysis

Objective:

Students will analyze their micropipetting accuracy, reflect on their technique, and understand how errors impact experimental outcomes.

Data Analysis:

• Interpreting Percent Error:
  • Compare the theoretical vs. actual mass of dispensed water.
  • Calculate and record the percent error for each micropipette size.
• Troubleshooting Pipetting Mistakes:
  • Identify any patterns in errors (e.g., consistent under-pipetting or over-pipetting).
  • Discuss factors that may have contributed to inaccuracies (e.g., improper technique, air bubbles, equipment calibration).
• Application to Real-World Scenarios:
  • How would micropipetting errors impact a PCR reaction?
  • What challenges might arise in a clinical or pharmaceutical lab due to poor pipetting accuracy?

Post-Lab Questions:

• Based on your percent error results, which micropipette size were you most accurate with? Why?
• What adjustments could you make to improve your pipetting accuracy?
• How does maintaining proper pipetting technique contribute to successful experiments in research and clinical settings?
• If your percent error was consistently high, what steps would you take to troubleshoot and correct it?
• What key takeaways did you learn from this lab that will help in future experiments?

By completing this pre-lab and post-lab reflection, students will develop a stronger foundation in micropipetting and its critical role in scientific research.