Week 1 Lab: Pipetting

1. Objectives

By the end of this lab, you will be able to:

• Identify the three most common micropipette types and their volume ranges
• Correctly set and read a volume on a micropipette
• Demonstrate proper aspiration and dispensing technique
• Understand common pipetting errors and how to avoid them

2. Background — What Is a Micropipette?

A micropipette (commonly just called a “pipette” in the lab) is a precision instrument used to measure and transfer very small volumes of liquid — typically between 0.1 µL and 1000 µL (1 mL). Accurate pipetting is one of the most fundamental skills in biology, chemistry, and biomedical research. Even small errors in volume measurement can ruin an experiment, skew results, or waste expensive reagents.

Volumes in the lab are measured in:

💡 Quick reference: 1 mL = 1,000 µL. A typical raindrop is ~50 µL. A grain of salt is ~60 nL.

3. Pipette Types and Volume Ranges

There are three standard micropipettes you will use in this course. Each is color-coded and designed for a specific volume range. Never exceed the maximum or go below the minimum volume — this damages the internal piston mechanism.

⚠️ Rule: Always choose the pipette whose range most closely fits your target volume. Using a P1000 to measure 5 µL will be wildly inaccurate.

4. Parts of a Micropipette

         ┌────────────────┐
         │   Thumb Knob   │  ← Push down to aspirate / dispense
         │   (Plunger)    │
         └───────┬────────┘
                 │
         ┌───────┴────────┐
         │  Volume        │  ← Twist to set volume (DO NOT exceed range)
         │  Adjustment    │
         │  Dial          │
         └───────┬────────┘
                 │
         ┌───────┴────────┐
         │  Volume        │  ← 3-digit display window
         │  Display       │     (read top to bottom)
         │  Window        │
         └───────┬────────┘
                 │
         ┌───────┴────────┐
         │   Barrel /     │
         │   Body         │
         └───────┬────────┘
                 │
         ┌───────┴────────┐
         │  Tip Ejector   │  ← Press to eject used tip (never touch used tips)
         │  Button        │
         └───────┬────────┘
                 │
         ┌───────┴────────┐
         │  Tip Cone /    │  ← Where disposable tip attaches
         │  Shaft         │
         └───────┬────────┘
                 │
            [  Tip  ]        ← Disposable plastic tip (changes every use!)

5. Reading the Volume Display

The volume display has three digits read from top to bottom. How you interpret those digits depends on which pipette you are using.

P20 (range: 0.5 – 20 µL)

  ┌───┐
  │ 1 │   ← tens digit (µL)
  │ 5 │   ← ones digit (µL)
  │ 0 │   ← tenths digit (µL)
  └───┘
  = 15.0 µL

P200 (range: 20 – 200 µL)

  ┌───┐
  │ 1 │   ← hundreds digit (µL)
  │ 5 │   ← tens digit (µL)
  │ 0 │   ← ones digit (µL)
  └───┘
  = 150 µL

P1000 (range: 200 – 1000 µL)

  ┌───┐
  │ 1 │   ← thousands digit (µL)  
  │ 0 │   ← hundreds digit (µL)
  │ 0 │   ← tens digit (µL)
  └───┘
  = 1000 µL = 1.0 mL

6. Step-by-Step Pipetting Procedure

Step 1 — Set the Volume

Turn the volume adjustment dial to your desired volume.

• Turn clockwise to decrease volume
• Turn counter-clockwise to increase volume
• Never twist past the maximum — you will damage the piston

Step 2 — Attach a Tip

Press the tip cone firmly into a fresh pipette tip in the tip box. Give it a slight twist or firm push to create an airtight seal. Never touch the tip with your fingers after attachment — this contaminates your sample.

Step 3 — Pre-Wet the Tip (for accuracy)

Before aspirating your actual sample, aspirate and expel the liquid 2–3 times to wet the inner walls of the tip. This reduces evaporation error, especially important for small volumes (<10 µL).

Step 4 — Aspirate (Draw Up Liquid)

1. Hold the pipette vertically (or no more than 20° from vertical)
2. Press the plunger down to the first stop (you will feel resistance — do NOT push to the second stop yet)
3. Submerge the tip 2–3 mm into the liquid (P20/P200) or 3–6 mm (P1000)
4. Slowly and smoothly release the plunger — liquid will draw up into the tip
5. Wait 1–2 seconds, then withdraw the tip from the liquid by sliding it along the container wall

⚠️ Releasing too fast creates bubbles and inaccurate volumes. Speed matters!

Step 5 — Check for Bubbles

Hold the tip up to the light. If you see a bubble, expel the liquid and re-aspirate. Bubbles displace liquid and reduce your actual volume.

Step 6 — Dispense Liquid

1. Touch the tip to the inner wall of the destination tube or well at a slight angle
2. Press the plunger slowly to the first stop — this expels the set volume
3. Then press to the second stop (blow-out position) — this expels any remaining droplet
4. Keep the plunger depressed while withdrawing the tip from the container
5. Release the plunger slowly after the tip has cleared the liquid

Step 7 — Eject the Tip

Press the tip ejector button firmly over a waste container. Never remove used tips by hand — tips may be contaminated with biological or chemical material.

7. Common Pipetting Mistakes & How to Avoid Them

8. Accuracy vs. Precision

These two terms are distinct and both matter in pipetting:

• Accuracy — how close your measured volume is to the true intended volume
• Precision — how reproducible your measurements are across repeated trials

        Accurate &        Precise but         Neither
        Precise           Not Accurate        (Random)
        
          [X]              [ ][ ]              [ X ]
          [X]              [ ][ ]X           X [   ]
          [X]              [ ][ ]            [  X  ]
        ← target →       ← target →        ← target →

A pipette can be precise but inaccurate if it is mis-calibrated. Always check calibration before critical experiments.

9. Gravimetric Accuracy Test (Optional Practice Exercise)

You can test your pipetting accuracy by weighing water. Since water has a density of 1.00 g/mL, 100 µL of water should weigh exactly 0.100 g.

Protocol:

1. Tare (zero) an analytical balance with a microcentrifuge tube
2. Using a P200, set to 100 µL
3. Aspirate distilled water and dispense into the tube
4. Record the mass
5. Repeat 5 times and calculate the mean and % error

Data Table:

% Error formula:

% Error = |Measured − Expected| / Expected × 100

✅ Good pipetting: % error < 2% for P200 at 100 µL
⚠️ Acceptable: % error 2–5%
❌ Needs improvement: % error > 5%

10. Safety and Disposal

• Always use a new tip for each new liquid or sample
• Never pipette corrosive acids, bases, or organic solvents with a standard micropipette — use a repeat pipettor or serological pipette
• Dispose of used tips in the biohazard waste bin if biological material was handled, or regular waste otherwise
• If liquid enters the barrel of the pipette, stop immediately and notify your instructor — the piston must be cleaned and re-calibrated