biotechnology 3 unit 13 study guides

Key Lab Equipment and Instruments

• Centrifuges separate materials based on density by spinning at high speeds
  • Microcentrifuges used for small volumes (1.5-2 mL tubes)
  • Larger centrifuges accommodate 15-50 mL tubes
• Micropipettes accurately measure and transfer small liquid volumes
  • Single-channel pipettes dispense one volume at a time
  • Multi-channel pipettes dispense multiple equal volumes simultaneously
• Spectrophotometers measure light absorption to determine sample concentration
  • UV-Vis spectrophotometers use ultraviolet and visible light
  • Nanodrop spectrophotometers require very small sample volumes (1-2 µL)
• Thermocyclers (PCR machines) amplify DNA through temperature cycling
• Gel electrophoresis separates DNA, RNA, or proteins based on size and charge
  • Agarose gels commonly used for DNA and RNA
  • Polyacrylamide gels used for proteins
• Incubators maintain optimal temperature and conditions for cell growth
• Biosafety cabinets provide sterile environment for cell culture and sensitive experiments

Safety Protocols and Best Practices

• Always wear appropriate personal protective equipment (PPE)
  • Lab coat, gloves, and safety glasses protect from hazards
  • Change gloves frequently to avoid contamination
• Handle biological materials and chemicals with caution
  • Use biosafety cabinets when working with infectious agents
  • Properly label and store hazardous materials
• Maintain a clean and organized workspace
  • Disinfect surfaces before and after experiments
  • Keep personal items separate from lab areas
• Follow proper waste disposal guidelines
  • Dispose of biohazardous waste in designated containers
  • Properly label and segregate chemical waste
• Be aware of emergency procedures and equipment locations
  • Know the location of fire extinguishers, eye wash stations, and safety showers
  • Familiarize yourself with evacuation routes and emergency protocols
• Report any accidents, injuries, or safety concerns immediately
• Attend safety training and stay updated on lab-specific protocols

Basic Lab Techniques

• Pipetting ensures accurate and precise liquid handling
  • Use the correct pipette for the volume range needed
  • Calibrate pipettes regularly to maintain accuracy
• Aseptic technique prevents contamination in microbiological work
  • Sterilize tools and surfaces using heat, chemicals, or filtration
  • Minimize exposure of sterile items to open air
• Solution preparation involves accurately weighing and dissolving reagents
  • Use analytical balances for precise measurements
  • Consider solubility and stability of reagents when preparing solutions
• pH measurement and adjustment is critical for many biological processes
  • Use pH meters or pH paper to determine the pH of solutions
  • Adjust pH using acids or bases as needed
• Centrifugation separates components based on size and density
  • Select appropriate centrifuge speed and time based on application
  • Balance tubes to ensure even spinning and prevent damage
• Spectrophotometry quantifies sample concentration based on light absorption
  • Create a standard curve using known concentrations
  • Blank the spectrophotometer with the solution used to dilute samples
• Microscopy allows visualization of small structures and organisms
  • Brightfield microscopy uses visible light to illuminate samples
  • Fluorescence microscopy detects fluorescently labeled molecules

Sample Preparation and Handling

• Sample collection and storage methods vary based on the type of sample
  • Use sterile containers and aseptic technique to prevent contamination
  • Store samples at appropriate temperature (room temp, 4°C, -20°C, -80°C)
• Cell lysis breaks open cells to release their contents
  • Mechanical methods include sonication and bead beating
  • Chemical methods use detergents or enzymes to disrupt cell membranes
• Protein extraction isolates proteins from cells or tissues
  • Denaturing conditions (heat, detergents) disrupt protein structure
  • Protease inhibitors prevent protein degradation during extraction
• Nucleic acid extraction purifies DNA or RNA from samples
  • Phenol-chloroform extraction separates nucleic acids from proteins and lipids
  • Solid-phase extraction uses silica columns to bind and elute nucleic acids
• Sample concentration may be necessary for downstream applications
  • Centrifugal concentrators remove solvent to increase sample concentration
  • Precipitation with ethanol or isopropanol concentrates nucleic acids
• Quantification ensures sufficient sample for analysis
  • Spectrophotometry measures nucleic acid and protein concentration
  • Fluorometric assays (Qubit) provide more sensitive quantification
• Quality control checks assess sample purity and integrity
  • Gel electrophoresis visualizes nucleic acid or protein size and degradation
  • Absorbance ratios indicate protein (A280) or nucleic acid (A260/A280) purity

Data Collection and Analysis

• Experimental design should include appropriate controls
  • Positive controls confirm the experimental system is working
  • Negative controls detect background noise or contamination
• Replicates increase the reliability and reproducibility of results
  • Technical replicates assess variability within an experiment
  • Biological replicates account for biological variation between samples
• Data organization is essential for efficient analysis
  • Use lab notebooks or electronic lab information management systems (LIMS)
  • Include metadata (date, experimenter, sample info) for each dataset
• Statistical analysis determines the significance of results
  • Calculate mean and standard deviation to summarize data
  • Use t-tests or ANOVA to compare means between groups
• Data visualization helps identify trends and communicate findings
  • Create graphs (bar, line, scatter) to display data
  • Use appropriate scales and labels for clarity
• Interpretation of results should consider limitations and potential sources of error
  • Assess the quality and reliability of data
  • Discuss findings in the context of previous research and biological significance
• Data storage and sharing enable collaboration and reproducibility
  • Back up data regularly to prevent loss
  • Use secure cloud storage or institutional servers for long-term storage

Troubleshooting Common Issues

• Contamination can lead to false results or failed experiments
  • Identify the source (reagents, samples, environment) and replace as needed
  • Implement stricter aseptic technique and use fresh sterile supplies
• Inconsistent results may indicate problems with technique or reagents
  • Verify proper calibration and functioning of equipment
  • Check the quality and expiration dates of reagents
• Low yield or poor quality of extracted molecules can hinder downstream applications
  • Optimize lysis and extraction protocols for specific sample types
  • Ensure proper sample storage and handling to prevent degradation
• Equipment malfunction can cause delays and inaccurate results
  • Perform regular maintenance and calibration of instruments
  • Contact technical support or repair services for persistent issues
• Unexpected or inconsistent data should be carefully examined
  • Review experimental design and controls for potential sources of error
  • Repeat the experiment to confirm the results
• Troubleshooting requires systematic problem-solving skills
  • Break down the issue into smaller components
  • Test one variable at a time to isolate the cause
• Consulting with colleagues or experts can provide valuable insights
  • Discuss the problem and potential solutions with lab members
  • Reach out to other researchers or technical support for advice

Advanced Biotech Procedures

• Polymerase Chain Reaction (PCR) amplifies specific DNA sequences
  • Design primers that flank the target sequence
  • Optimize annealing temperature and cycle number for efficient amplification
• DNA sequencing determines the order of nucleotides in a DNA molecule
  • Sanger sequencing uses dideoxy chain termination
  • Next-generation sequencing (NGS) enables high-throughput parallel sequencing
• Cloning inserts DNA fragments into a vector for propagation
  • Restriction enzymes and ligase are used for traditional cloning
  • Gibson assembly or Golden Gate assembly enable seamless cloning
• Genetic engineering modifies the genetic material of an organism
  • CRISPR-Cas9 allows precise gene editing and regulation
  • Plasmid vectors introduce foreign DNA into cells for expression
• Cell culture involves growing cells in a controlled laboratory environment
  • Maintain proper sterility and growth conditions for each cell line
  • Regularly passage cells to prevent overcrowding and maintain viability
• Protein expression systems produce recombinant proteins
  • Bacterial (E. coli), yeast, or mammalian cells are common expression hosts
  • Optimize codon usage, induction conditions, and purification methods
• Immunoassays detect and quantify specific proteins
  • ELISA (enzyme-linked immunosorbent assay) is widely used for antibody-based detection
  • Western blotting separates proteins by size and detects with specific antibodies

Documentation and Reporting

• Lab notebooks serve as a permanent record of experiments
  • Use bound notebooks with numbered pages and write in permanent ink
  • Record all relevant information (date, objective, methods, results, conclusions)
• Standard operating procedures (SOPs) ensure consistency and reproducibility
  • Develop detailed, step-by-step instructions for common lab procedures
  • Regularly review and update SOPs to reflect current best practices
• Experimental reports communicate findings to others
  • Include an abstract, introduction, methods, results, and discussion sections
  • Present data clearly using figures and tables with appropriate legends
• Inventory management tracks the supplies and reagents used in the lab
  • Use a centralized system to monitor stock levels and expiration dates
  • Implement a labeling and organization scheme for easy retrieval
• Collaboration and data sharing facilitate scientific progress
  • Use version control (e.g., Git) to track changes and contributions
  • Deposit data in public repositories (GenBank, GEO) for community access
• Intellectual property and confidentiality considerations may apply
  • Consult with technology transfer offices for guidance on patents and licensing
  • Use non-disclosure agreements (NDAs) when sharing sensitive information
• Scientific integrity and responsible conduct of research are paramount
  • Accurately represent data and methods in publications and presentations
  • Disclose potential conflicts of interest and adhere to ethical guidelines