1Fully automatic PCR machinePre-operation preparation
Preparation of reagents and consumables
Reagents: including DNA template, specific primers (forward and reverse), dNTPs (four types of deoxyribonucleotides), high-temperature resistant DNA polymerase (such as Taq enzyme), buffer solution, and Mg ² ⁺ ion solution. All reagents must be completely melted before mixing to avoid concentration errors.
Consumables: Use disposable sterile PCR tubes or 96 well plates to ensure good sealing and prevent cross contamination.
Partition operation: Complete system preparation in the reagent preparation area (ultra clean workbench or biosafety cabinet), wear gloves and avoid speaking during operation to reduce aerosol pollution.
Instrument inspection
Confirm that the PCR instrument is placed on a level and sturdy platform, with matching power supply voltage and good grounding.
Check if the sample tank is clean to avoid residue affecting temperature conduction.
Regularly clean the sample tank with soapy water, and prohibit the use of strong alkali, high concentration alcohol, or organic solvents.
2、 Preparation of reaction system
Mixed reagents
Add the DNA template, primers, dNTPs, DNA polymerase, buffer, and Mg ² ⁺ solution in a sterile centrifuge tube in proportion, and add an appropriate amount of double distilled water to the final volume (recommended 15-50 μ L).
Key parameters:
DNA template quality: Avoid degradation or inhibitor contamination, otherwise it will affect amplification efficiency.
Primer design: Ensure specificity, avoid dimers or self complementary structures, and the annealing temperature should match the Tm value of the primer.
Mg ² ⁺ concentration: Excessive or insufficient concentration can inhibit enzyme activity, and should be adjusted according to the reagent instructions.
Packaging system
Quickly transfer the mixture into PCR tubes, cover the tube tightly or with a specialized membrane, and centrifuge briefly (1000-2000rpm, 1 minute) to allow the liquid to settle to the bottom of the tube.
3、 PCR instrument program settings
Temperature program parameters
Initial denaturation: 94-98 ℃, 20-30 seconds, to completely separate the DNA double strand.
Cyclic denaturation: Maintain single chain state at 94-98 ℃ for 20-30 seconds.
Annealing: 50-65 ℃, 20-40 seconds, primer and template specific binding (annealing temperature needs to be optimized by gradient PCR).
Extension: 72 ℃, 1 minute/kb DNA fragment, Taq enzyme synthesizes new chains.
Cycle number: 20-40 times, adjusted according to the abundance of the target fragment.
Final extension: 72 ℃, 5-10 minutes, ensure complete extension of all fragments.
Storage temperature: 16 ℃, 2 minutes (to avoid excessive wear of the compressor, it is forbidden to set it to 4 ℃ for indefinite storage).
Advanced feature settings
Temperature gradient: Set a temperature gradient (such as 50-65 ℃) during the annealing step, and test multiple annealing temperatures simultaneously to optimize experimental conditions.
Ramp Rate: Adjust the heating/cooling rate (e.g. 3 ℃/s) to shorten the reaction time.
Increment: The temperature or time increases after each cycle (such as heating up by 0.1 ℃ per cycle), suitable for special experiments such as Touchdown PCR.
Program saving and running
After entering the program name and saving, place the PCR tube into the sample slot, close the hot cap, and tighten it.
Start the program and monitor the temperature and time in real-time through the screen during operation. It supports pausing, skipping steps, or terminating the program.
4、 Post experimental processing
product analysis
Take out the PCR tube immediately after reaction, and analyze the product size and concentration through agarose gel electrophoresis or fluorescence detection (such as SYBR Green).
If fluorescence labeling method is used, it is necessary to ensure that the instrument parameters (such as excitation wavelength, gain value) match the reagents and avoid background signal interference.
instrument maintenance
Before turning off the instrument, allow the heating module to cool naturally to room temperature to prevent thermal stress damage.
Regularly calibrate temperature sensors to ensure temperature accuracy (such as using standard melting point calibrators).
Clean the sample tank and hot cover, check if the sealing ring is aging, and replace any damaged parts in a timely manner.
5Fully automatic PCR machinePrecautions
Pollution prevention and control
The entire experiment follows the principle of "no nucleic acid", using specialized pipettes, gun tips, and work clothes to avoid cross contamination.
Establish negative controls (without templates) and positive controls to monitor the reliability of the experimental system.
safe operation
Avoid opening the hot cover during PCR machine operation to prevent high-temperature burns or sample evaporation.
Do not use corrosive liquids to clean instruments to prevent damage to electronic components.
troubleshooting
No amplification products: Check template quality, primer design, or program parameters (such as high annealing temperature).
Non specific bands: Optimize annealing temperature or increase extension time to reduce dimer formation.
Temperature fluctuations: Contact the engineer to calibrate the instrument and ensure that the temperature uniformity meets the standard (such as ± 0.2 ℃).