Listen to Theory Module
Listen to this module while following along with the text below, or download for offline study.
Overview
This module covers essential laboratory practices that form the foundation of all molecular biology work.
Understanding these fundamentals ensures safe, accurate, and reproducible experimental results throughout
your career in molecular entomology and DNA barcoding.
Key Topics Covered
1. Laboratory Safety
- Personal Protective Equipment (PPE): Proper use of gloves, lab coats, safety goggles, and closed-toe shoes
- Chemical Hazards: Understanding MSDS/SDS, GHS signal words, H-codes and P-codes
- Common Reagents: Safe handling of guanidinium thiocyanate, Proteinase K, ethanol, and UV radiation
- First Aid: Proper response to chemical exposure and when to seek medical attention
2. Workspace Organization and Contamination Prevention
- Workspace Preparation: Pre- and post-work cleaning protocols with 70% ethanol
- Why 70% Ethanol? Understanding the molecular basis for optimal disinfection
- Workflow Organization: Clean-to-dirty workspace setup and directional work patterns
- Contamination Sources: Human DNA, aerosols, surfaces, and PCR amplicons
3. Accurate Pipetting Techniques
- Micropipette Mechanics: Air displacement, two-stop system, and piston operation
- Sources of Error: Temperature, viscosity, surface tension, and technique variability
- Proper Technique: Pre-wetting, vertical position, immersion depth, and controlled aspiration
- Volume-Specific Considerations: Small (1-10 µL), medium (10-200 µL), and large volumes (200-1000 µL)
4. Solution Preparation and Dilutions
- Stock vs. Working Solutions: Concentration, storage, and cost efficiency
- Dilution Calculations: C1V1 = C2V2 equation and practical applications
- Serial Dilutions: Creating standard curves and systematic concentration reduction
- Aliquoting: Preventing freeze-thaw degradation and contamination of stocks
5. DNA Stability and Preservation
- Degradation Factors: Physical (shear), chemical (pH, oxidation), and enzymatic (DNases)
- High Molecular Weight DNA: Importance for long-read sequencing and genome assembly
- Storage Conditions: Temperature, buffer choice (TE vs. water), and freeze-thaw cycles
- Handling Practices: Avoiding vortexing, using filter tips, and gentle mixing
6. Aseptic Technique
- Environmental Controls: Clean workspace, minimal air currents, UV decontamination
- Personal Practices: Hand washing, PPE, and glove changes
- Working Technique: Tube handling, cap management, and minimizing exposure time
- Contamination Sources: Skin cells, aerosols, surfaces, and reagents
7. Laboratory Notebooks
- Scientific Purpose: Reproducibility, troubleshooting, IP protection, and data integrity
- Required Elements: Header, materials, methods, results, analysis, and conclusions
- Format and Organization: Permanent ink, dated entries, numbered pages, table of contents
- Data Recording: Immediate entry, units, calculations, and significant figures
Critical Safety Reminders
Always remember:
- Flush chemical exposures with water for 15 minutes minimum
- Report ALL chemical incidents to your instructor immediately
- Never pipette by mouth - always use mechanical pipetting devices
- Keep lab coat buttoned/closed at all times during lab work
- 70% ethanol requires 30 seconds contact time for effective decontamination
- Change gloves between procedures and whenever contaminated
Best Practices for Success
- Pre-wet pipette tips 2-3 times for small volumes to improve accuracy
- Use filter tips for all DNA work to prevent aerosol contamination
- Label tubes before adding samples to prevent mix-ups
- Work in one direction (clean to dirty) to minimize cross-contamination
- Aliquot expensive reagents to prevent freeze-thaw degradation
- Store DNA in TE buffer, not water, for long-term stability
- Include negative controls in all experiments to detect contamination
- Record data immediately in your lab notebook - don't trust your memory
Why These Fundamentals Matter
Molecular biology relies on detecting specific DNA sequences, often at extremely low concentrations
(nanogram levels). Even trace contamination can cause false results that invalidate entire experiments.
The practices covered in this module aren't arbitrary rules - they're evidence-based protocols developed
to ensure:
- Your Safety: Preventing exposure to hazardous chemicals and UV radiation
- Data Quality: Minimizing contamination and technical errors
- Reproducibility: Allowing others to replicate your experiments
- Efficiency: Preventing wasted time and resources on failed experiments
- Professional Development: Building skills you'll use throughout your career
Applying These Skills
Throughout this course, you will apply these fundamental skills in every laboratory module:
- DNA Extraction: Aseptic technique, accurate pipetting, contamination prevention
- PCR Setup: Serial dilutions, solution preparation, aerosol control
- Gel Electrophoresis: Solution preparation, safety protocols, data recording
- Sequencing Preparation: High molecular weight DNA handling, quality control
- Data Analysis: Laboratory notebook documentation, reproducibility
Master these fundamentals now, and every subsequent experiment will be more successful.
Scientific Literature References
This module is supported by peer-reviewed scientific literature on laboratory safety,
contamination prevention, and best practices in molecular biology.
View Complete References →