Instrumentation Prelim Week 2 PDF

University of the Immaculate Conception Fr. Selga Street, Davao City College of Medical and Biological Sciences Medical Technology/Medical Laboratory Science INSTRUMENTATION P...

University of the Immaculate Conception Fr. Selga Street, Davao City College of Medical and Biological Sciences Medical Technology/Medical Laboratory Science INSTRUMENTATION Prelim| Week 2 Molecular Biology and Diagnostics | Laboratory | 2024-2025 Learning Objectives Week 2 Molecular Biology At the end of this exercise, the student can: a. identify and describe the function of key instruments used in molecular biology, including PCR machines, gel electrophoresis, spectrophotometers, centrifuge and many more. b. explain how these instruments are applied in the analysis of nucleic acids and proteins; and c. understand the safety protocols associated with use of molecular biology instruments Molecular Biology and Diagnostics | Laboratory | 2024-2025  Week 2 Common Instruments in Molecular Biology Molecular Biology Microcentrifuge Gel Electrophoresis qPCR Vortex Ultra-Low Temperature Freezer Molecular Biology and Diagnostics | Laboratory | 2024-2025  Week 2 Common Instruments in Molecular Biology Molecular Biology Spectrophotometer PCR Clean bench BSC Class II Molecular Biology and Diagnostics | Laboratory | 2024-2025  Week 2 MICROCENTRIFUGE Molecular Biology ➔Used to separate cellular components from nucleic acids ➔Typically handle small volumes, from 0.2 to 2 mL, which are ideal for molecular biology experiments ➔Can achieve high rotational speed of 15,000-20,000 RPM Molecular Biology and Diagnostics | Laboratory | 2024-2025  Week 2 MICROCENTRIFUGE | Reminders Molecular Biology ➔ Always ensure that tubes placed inside are balanced (equal weight and placed symmetrically) ➔ NEVER attempt to slow or stop the rotor by hand ➔ Do not exceed the maximum rated speed of the rotor in use ➔ Regularly inspect and maintain microcentrifuge according to manufacturer’s instruction Molecular Biology and Diagnostics | Laboratory | 2024-2025  Week 2 ULTRA-LOW TEMPERATURE FREEZER Molecular Biology ➔Used to store DNA, RNA, and other nucleic acids (at -40 to -80 degrees Celsius) for long periods without degradation. Thus, maintaining sample integrity. ➔Extreme cold prevents growth of bacteria and other contaminants, ensuring purity of stored biological samples. ➔Some freezers utilize liquid nitrogen (LN₂) Molecular Biology and Diagnostics | Laboratory | 2024-2025  Week 2 ULTRA-LOW TEMPERATURE FREEZER | Reminders Molecular Biology ➔ Properly label samples, including date and content to avoid misplacement or confusion ➔ NEVER TOUCH frozen items with bare hands to prevent frostbite or skin damage. ➔ Ensure freezer door is closed tightly after use. Avoid opening door for extended time periods. ➔ Keep the freezer organized and avoid overcrowding ➔ Ensure proper ventilation ➔ Monitor freezer’s temperature regularly Molecular Biology and Diagnostics | Laboratory | 2024-2025  Week 2 ULTRA-LOW TEMPERATURE FREEZER | Reminders Molecular Biology Maintenance Schedule Item Interval Defrost Defrost freezer once per year whenever the ice build exceeds 3/8 “ (0.95 cm) Gasket Periodically check the gaskets around the door for punctures or tears. Periodically clean the ice build-up around it. Filter Clean the condenser filter(s) every 2 to 3 months Condenser Clean every 6 months, more often if the lab area is dusty Battery Replace battery as indicated by system alarms or as necessary per individual status evaluation. Check battery connections regularly. Annual battery replacement is recommended. Molecular Biology and Diagnostics | Laboratory | 2024-2025  Week 2 GEL ELECTROPHORESIS Molecular Biology ➔Used to isolates DNA or RNA fragments according to their size ➔Also used to assess the quality and integrity of nucleic acid or protein samples ➔Small fragments migrate faster through the gel matrix, while larger fragments GELATO move slowly. Molecular Biology and Diagnostics | Laboratory | 2024-2025  Week 2 GEL ELECTROPHORESIS Molecular Biology Molecular Biology and Diagnostics | Laboratory | 2024-2025  Week 2 GEL ELECTROPHORESIS Molecular Biology Molecular Biology and Diagnostics | Laboratory | 2024-2025  Week 2 GEL ELECTROPHORESIS Molecular Biology Molecular Biology and Diagnostics | Laboratory | 2024-2025  Week 2 GEL ELECTROPHORESIS | Reminders Molecular Biology ➔Handle chemicals carefully (e.g. ethidium bromide, agarose gel, etc.) ➔Ensure electrophoresis equipment is properly set up ➔Proper disposal of waste ➔AVOID CONTAMINATION ➔Ensure proper gel loading by performing proper pipetting technique Molecular Biology and Diagnostics | Laboratory | 2024-2025  Week 2 qPCR Molecular Biology ➔aka REAL-TIME PCR, used to measure and quantify the amount of specific nucleic acid present in a sample ➔Applied in the following: - Gene expression analysis - Pathogen detection and diagnosis - PCR cloning and recombinant DNA studies qTOWERiris - Mutation detection and many more… Molecular Biology and Diagnostics | Laboratory | 2024-2025  Week 2 qPCR Molecular Biology Molecular Biology and Diagnostics | Laboratory | 2024-2025  Week 2 qPCR | Reminders Molecular Biology ➔Wear personal protective equipment (PPE) ➔Always use clean pipettes, tips, and containers to avoid DNA contamination ➔If working with RNA samples, ensure equipment and reagents are RNase-free ➔Proper temperature management ➔Include negative controls in PCR reactions to check for contamination Molecular Biology and Diagnostics | Laboratory | 2024-2025  Week 2 VORTEX MIXER Molecular Biology ➔Used to rapidly mix small volumes of liquid samples. ➔Primary function is to create a vortex or swirling motion to ensure thorough mixing of sample contents ➔Has adjustable speed of mixing depending of solution viscosity Molecular Biology and Diagnostics | Laboratory | 2024-2025  Week 2 VORTEX MIXER | Reminders Molecular Biology ➔ Ensure the motor is running smoothly. If vortex makes unusual noises or vibrates excessively, stop using it and report for maintenance ➔ Properly secure tubes ➔ NEVER overload vortex with too many tubes ➔ Set the speed correctly ➔ Must be places on a stable, flat surface to prevent moving or falling ➔ Do NOT press down forcefully while using Molecular Biology and Diagnostics | Laboratory | 2024-2025  Week 2 CLEAN BENCH Molecular Biology ➔aka LAMINAR FLOW HOOD or LAMINAR FLOW CABINET; used to provide a sterile and controlled environment (aseptic) for conducting experiments to prevent contamination ➔Filtered using HEPA filters to remove contaminants ➔Maintain positive air pressure Molecular Biology and Diagnostics | Laboratory | 2024-2025  Week 2 CLEAN BENCH Molecular Biology Molecular Biology and Diagnostics | Laboratory | 2024-2025  Week 2 CLEAN BENCH Molecular Biology Clean bench structure: ➔Plenum box - most important system to ensure the equipment performance ➔Base - most important components to support working chamber Has 3 parts: left base support, rear base support, & right base support ➔Working chamber -consists of working framework, stainless steel cabinet surface Molecular Biology and Diagnostics | Laboratory | 2024-2025  Week 2 CLEAN BENCH Molecular Biology Clean bench structure: ➔HEPA filter - filter efficiency of 99.9% which is used as the last filter equipment during air purification ➔Roughing filter - used as pre-filter for HEPA filter ➔Fan - provides the source of power for the air flow in the ventilation circuit. ➔Electrical appliance Molecular Biology and Diagnostics | Laboratory | 2024-2025 CLEAN BENCH 2 Types of Clean Benches Vertical Laminar Flow Cabinet Horizontal Laminar Flow Cabinet Air flows from the top to the work Air flows from the back of the bench toward surface providing protection for the the user. Used for tasks where there is less sample risk of contamination. Molecular Biology and Diagnostics | Laboratory | 2024-2025  Week 2 CLEAN BENCH | Reminders Molecular Biology ➔Verify HEPA filters and check proper airflow ➔Ensure clean bench has been properly cleaned and decontaminated before use ➔Minimize clutter ➔Seal containers immediately to avoid contamination ➔Do NOT obstruct air vents ➔Use built-in UV lights for decontaminating surfaces between uses Molecular Biology and Diagnostics | Laboratory | 2024-2025  Week 2 CLEAN BENCH | Reminders Molecular Biology ➔Carry out maintenance every year or every 1,000 working hours, or every time cabinet is restarted ➔DAILY OR WEEKLY cleaning during use: Use medical alcohol for disinfection Use glass detergent to clean outer surface ➔Replace filters when dusts accumulate on HEPA filters (only authorized people trained by Haier Group) Molecular Biology and Diagnostics | Laboratory | 2024-2025  Week 2 SPECTROPHOTOMETER Molecular Biology ➔Used in measuring the concentration and purity of nucleic acids and proteins ➔Allows the measurement of very small sample volumes (as low as 1-2 uL) ➔ Based on the absorbance value using the Beer- Lambert Law PURITY = ~ 1.8 DNA and ~2.0 for RNA NO Nanodrop DNA/RNA absorbance = 260 nm cuvettes needed Molecular Biology and Diagnostics | Laboratory | 2024-2025  Week 2 SPECTROPHOTOMETER Molecular Biology Molecular Biology and Diagnostics | Laboratory | 2024-2025  Week 2 SPECTROPHOTOMETER Molecular Biology ➔AVOID direct contact with pedestal ➔Use proper pipetting techniques (avoid introducing air bubbles or contamination) ➔Use lint-free tissue or cloth and distilled water in cleaning pedestal after use ➔Check for proper and report any malfunctions or concerns ➔Monitor Purity rations (260/280 nm ratio) Molecular Biology and Diagnostics | Laboratory | 2024-2025  Week 2 PCR Molecular Biology ➔Used to amplify specific DNA sequences (producing millions of copies of a particular DNA segment) ➔Features rapid thermal cycling ➔Optimized for smaller reaction volumes ➔Useful for field diagnostics, forensic analysis, and environmental monitoring miniPCR Molecular Biology and Diagnostics | Laboratory | 2024-2025  Week 2 PCR Molecular Biology Molecular Biology and Diagnostics | Laboratory | 2024-2025  Week 2 PCR | Reminders Molecular Biology ➔NO overloading with more samples than it is designed to handle ➔Use sterile tools when adding or transferring samples into PCR tubes ➔Follow correct loading procedures ➔Do NOT open during amplification ➔Allow cooling before handling (thermal miniPCR block is hot and could cause burns Molecular Biology and Diagnostics | Laboratory | 2024-2025  Week 2 BIOSAFETY CABINET CLASS II Molecular Biology ➔Provides a controlled environment for handling biological materials while also protecting the user, the environment, and the sample ➔Installed with HEPA filters ➔Different types: Type A1, Type A2, Type B1, and Type B2 Molecular Biology and Diagnostics | Laboratory | 2024-2025  Week 2 BIOSAFETY CABINET CLASS II Molecular Biology Molecular Biology and Diagnostics | Laboratory | 2024-2025  Week 2 BIOSAFETY CABINET CLASS II Molecular Biology Molecular Biology and Diagnostics | Laboratory | 2024-2025  Week 2 BIOSAFETY CABINET CLASS II Molecular Biology Molecular Biology and Diagnostics | Laboratory | 2024-2025  Week 2 BIOSAFETY CABINET CLASS II | Reminders Molecular Biology ➔Wear appropriate PPE ➔Adjust sash to recommended height (6-8 in.) ➔Avoid blocking airflow ➔Disinfect BSC before and after working ➔Avoid overfilling the workspace ➔Allow BSC to run for at least 5-10 minutes to stabilize airflow ➔Have annual certification and regular HEPA filter checks Molecular Biology and Diagnostics | Laboratory | 2024-2025  University of the Immaculate Conception Fr. Selga Street, Davao City College of Medical and Biological Sciences Medical Technology/Medical Laboratory Science INSTRUMENTATION Prelim| Week 2 Molecular Biology and Diagnostics | Laboratory | 2024-2025