Lecture Notes: Introduction to Tools Used in Biochemistry Experiments

Overview

• Objective: Familiarize students with basic principles and proper usage of tools in biochemistry experiments.
• Tools Covered:
  1. Micropipette
  2. pH Meter
  3. Centrifuge
  4. SDS-PAGE Electroporator

Micropipette

• Purpose: Transfer small liquid volumes (<1 ml) on a microliter scale.
• Types:
  • P1000: Max 1000 µl (accuracy: 200-1000 µl)
  • P200: Max 200 µl (accuracy: 20-200 µl)
  • P20: Max 20 µl (accuracy: 0.5-20 µl)
• Components:
  1. Plunger (ejects liquid; displays scale)
  2. Tip Eject Button (removes tip)
  3. Volume Adjustment Dial (changes liquid volume)
  4. Volume Readout (displays liquid volume)
  5. Tip Eject Shaft (assists tip ejection)
  6. Tip Attachment (holds the tip)
• Usage Steps:
  1. Press plunger to initial resistance; dip tip into liquid (avoid container bottom).
  2. Release plunger slowly; remove tip from liquid.
  3. Transfer liquid to new container; press plunger to eject.
  4. Release plunger slowly; eject tip.
• Cautions:
  • Do not exceed volume limits.
  • Handle plunger stop points carefully.
  • Maintain sterility; avoid contamination.

pH Meter

• Purpose: Measure pH (acidity/alkalinity) of solutions.
• Components:
  • Electrode (sensitive to H+ ions)
  • Electronic meter (displays pH reading)
• Calibration:
  • Use 2-3 buffer solutions (e.g., pH 4, 7, 10) for range coverage.

Centrifuge

• Purpose: Separate particles based on size, density, and speed.
• Types:
  • Swing-out rotor: Tubes rotate freely; horizontal orientation.
  • Fixed-angle rotor: Tubes fixed; faster due to less air friction.
• Process:
  • Pre-centrifugation: Evenly dispersed particles.
  • Post-centrifugation: Supernatant (less dense) & Pellet (more dense)
• Speed & Force:
  • Microcentrifuge: Max 12,000 rpm
  • High-velocity: Max 25,000 rpm
  • Ultracentrifuge: Max 18,000 rpm
• Cautions:
  • Operate only when closed and balanced.
  • Fill tubes 2/3 or 3/4 full.
  • Ensure motor stops before opening.

SDS-PAGE Electroporator

• Purpose: Separate proteins by molecular weight via electrophoresis.
• Principle: Charged molecules move towards opposite electrodes; smaller migrate faster.
• Components & Setup:
  1. Casting Stand & Frame (gel molding)
  2. Plastic Comb (creates wells)
  3. Glass Plates with Spacer (holds gel)
  4. Sample Loading Guide (assists sample placement)
  5. Clamping Frame & Electrode (holds gel during electrophoresis)
• Process:
  1. Clean apparatus with ethanol.
  2. Assemble glass plates and conduct leak test.
  3. Prepare acrylamide solution; pour and let polymerize.
  4. Install gel in chamber; add buffer and load samples.

Conclusion

• Review key steps and precautions for each tool.
• Stay healthy and practice safe lab procedures.