Thermocycler standard operation,pcr.pptx

Thermocyler, Sop of thermocycler

1. Thermocycler ( Standard Operating Procedure) Dr. Anoop Kumar Junior Resident 2 nd year Department of Pharmacology and Therapeutics King George’s Medical University,Lucknow,[U.P., India] 226003 [email protected]

2. Content Introduction Principle Parts of the Instrument Sop of Thermocycler Applications Merits & Demerits Summary

3. Learning Objectives

4. Introduction A Thermocycler , also known as a PCR (Polymerase Chain Reaction) machine , is a laboratory instrument used to amplify specific DNA sequences through the process of Polymerase Chain Reaction (PCR) . Function: It enables rapid and precise replication of DNA by cycling through different temperature phases. Significance: PCR is a cornerstone technique in molecular biology, widely used in research, diagnostics, forensic science, and genetic studies.

5. Principle of Operation A thermocycler enables PCR (Polymerase Chain Reaction) by precisely controlling temperature cycles for DNA amplification. It automates the following key steps: Denaturation (94–98°C, 15–30 sec): High heat breaks hydrogen bonds, separating double-stranded DNA into single strands. Annealing (50–65°C, 15–60 sec): Primers bind to specific complementary DNA sequences, defining the target region for amplification. Extension (72°C, 30–60 sec per kb): Taq polymerase synthesizes new DNA strands by adding nucleotides (dNTPs) to the primer-bound template. Each cycle doubles DNA quantity , leading to exponential amplification over 25–40 cycles .

6. Number pad Enter key Standby Delete key Stop key Start key Lid Handle Softkeys Parts of Instrument

7. Heated lid Thermoblock Display Arrow keys Status lamp

8. Air vents Ethernet port Connection socket CAN in port CAN out port Eco and Term switch Power switch

9. Thermoblock PCR Tube

10. PCR Reagents DNA template Primers DNA polymerase dNTPs (nucleotides) Reaction buffer

11. Precautions Maintain a clear workspace around the device Wear gloves to prevent contamination of samples Avoid operating the lid without properly loaded samples Ensure lid sits flat against the top of the tubes Handle heated surfaces carefully to avoid burns Once the use of the PCR machine is finished , it should be turned off

12. Standard Operating Procedure Prepare samples with PCR reagents Load samples into PCR tubes or plates and place them in the thermoblock Close the heated lid to ensure proper sealing Set denaturation, annealing, and extension temperatures Input cycle numbers based on experimental needs Start the program using the Start Key Monitor the progress Switch off the instrument

13. Maintenance and Calibration Task Frequency Procedure Exterior Cleaning Weekly Wipe with a lint-free cloth and disinfectant. Avoid harsh chemicals. Thermoblock Cleaning After each use Clean with ethanol/isopropanol to remove residue and prevent contamination. Lid & Seal Check Weekly Inspect and clean the heated lid; ensure proper sealing to prevent evaporation. Vent & Fan Inspection Monthly Clear dust buildup to prevent overheating and ensure airflow. Temperature Calibration Every 6 months Use a temperature probe or calibration kit to verify accuracy. Adjust if needed. Lid Pressure Check Every 6 months Ensure uniform lid pressure for consistent heating across all wells.

14. Troubleshooting Issue Possible Cause Solution No Power / Won’t Turn On - Loose power cord - Blown fuse - Overheating shutdown - Check connections - Replace fuse - Allow cooldown & restart Temperature Errors - Lid not sealed - Sensor malfunction - Blocked air vents - Ensure lid is closed - Run diagnostic test - Clean vents Poor or No PCR Amplification - Incorrect cycle settings - Contaminated reagents - Primer issues - Verify PCR program - Use fresh reagents - Check primer design

15. Issue Possible Cause Solution Sample Evaporation - Loose lid pressure - Tube/plate not sealed - Heated lid malfunction - Adjust lid pressure - Use PCR-certified tubes - Check lid settings Software Malfunctions - Outdated firmware - Memory/storage issues - Incorrect programming - Update firmware - Clear old programs - Verify cycle settings Unusual Noise or Mechanical Issue - Loose internal parts - Fan/motor wear - Unstable placement - Contact support - Clean/inspect parts - Ensure stable surface

16. Applications of Thermocycler Medicine & Diagnostics: Detects genetic mutations, infectious diseases (COVID-19, HIV, TB), and cancer-related gene alterations. Forensic Science: Enables DNA fingerprinting, paternity testing, and disaster victim identification. Genetic Research: Supports genome comparison, gene expression analysis, and CRISPR-based modifications. Evolutionary Biology: Analyzes ancient DNA and traces phylogenetic relationships. Agriculture & Food Safety: Detects GMOs, plant pathogens, and aids in livestock genetic screening.

17. Merits of Thermocycler Precise thermal control ensures accurate and reproducible PCR results. Energy-efficient models reduce power consumption and operational costs. User-friendly interface with intuitive controls for easy operation. Programmable & automated for customized cycling and real-time monitoring. High throughput capability allows simultaneous processing of multiple samples. Fast and reliable DNA amplification with optimized heating and cooling rates. Compact and durable design for long-term, efficient performance.

18. Demerits of Thermocycler Error-prone DNA polymerase may cause mutations in amplified products. Highly susceptible to contamination , leading to false results. Expensive equipment with high initial and maintenance costs. Requires skilled operators for accurate setup and result interpretation.

19. Summary It is a lab device used for performing PCR to amplify DNA Creates multiple copies of specific DNA sequences The machine cycles through different temperatures to enable DNA denaturation, primer annealing, and DNA extension It has precise temperature control, adjustable cycles, and programmable settings Used in medicine, forensics, agriculture, and molecular biology Compact, easy to use, and essential for DNA-based experiments

20. Learning Objectives Achieved

21. References Eppendorf SE. Mastercycler ® Nexus Operating Manual. Hamburg: Eppendorf SE; 2021. Eppendorf Mastercycler Nexus X2 - PCR Thermal Cycler. Eppendorf AG Eppendorf Mastercycler Nexus Thermal Cycler. The Lab World Group

22. Questions

23. THANK YOU