Thermal Cyclers are laboratory apparatus that amplify segments of DNA using Polymerase Reaction (PCR). A thermal cycler is also known as a DNA Amplifier, PCR Machine, or Thermocycler. It consists of a thermal block with holes for inserting tubes, allowing PCR mixture reaction to occur. The cycle normally lowers or raises the block’s temperature in pre-programmed, discrete steps. A thermal cycler is important for a laboratory dealing in molecular biology and gene cloning. Thermal cyclers are available in various models and sizes. You can purchase larger or compact models depending on lab space.
A common Thermal Cycler contains the following components:
- A DNA template with a DNA region for amplification
- Two primers that complement the five primes of the DNA region
- Building blocks where the polymerase synthesize the DNA strand
- A DNA polymerase
- A buffer solution that provides an optimum environment for the stability of DNA polymerase
- Divalent and monovalent cations
Different Types of Thermal Cycler Calibration
ROI calibration maps the sample block and establishes the positions of the wells using the SDS software. When ROI calibration is performed, the SDS software will increase the fluorescence at each specific well of the reaction plate during the instrument operation. ROI calibration is performed every six months or when you want to verify the performance of your thermal cycler.
The SDS software measures the ambient fluorescence from the background electric signals, consumables, water inside the consumables, and sampling blocks when performing the background calibration. The background calibration helps the SDS software increase instrument precision by eliminating the background signals from the fluorescent samples. Background calibration is performed every month when installing an uncalibrated block or before performing a pure dye calibration.
Pure Dye Spectra Calibration
The purpose of the pure dye spectra calibration is to distinguish each fluorescent dye used in your instrument operation. The SDS software processes each set of pure dye standards using a set of spectral data it receives at each run. Pure dye spectra calibration is performed every six months or before installing an uncalibrated block.
Instrument Verification Run
Instrument verification run calibration ensures that the thermal cycle can calculate quantities of two unknowns by providing a standard curve. Instrument verification run calibration is done every six months or when you want to verify your thermal cycler performance.
How to Calibrate a Thermal Cycler
Calibration By Imitation of The PCR Process
It is the best method to calibrate your thermal cycler. The calibration method mimics the normal PCR process, thus providing precise results. When conducting this calibration method, keep the process at a real temperature using a particular reaction mixture in your thermal cycler tube. The process must be carried while mimicking a PCR protocol from a particular instrument. Use a variety of mix combinations and repeat the process to establish consistency using the PCR protocol. Collect different variables during the experiment by conducting your exercise using the thermal cycler’s temperature range under calibration. When you copy the PCR process at a particular temperature, you will find the uncertainty range of your thermal cycler without inactivating the polymerase.
Representative Process Calibration
The representative calibration process uses the usual standard measurement of the PCR process. The calibration method uses both the static and the dynamic aspects of the standard PCR. When conducting this calibration process, you will be able to solve problems that are hard to detect by the naked eye. The process calibrates your thermal cycler by checking the temperature during hybridization, elongation, and denaturation.
Calibration According to International Standards
It is the perfect method to calibrate a thermal cycler when working in a metrology setup. The calibration process by international standards is carried out in specific conditions. The primary advantage of this process is that you will be able to get traceability of your calibration by comparing them with the reference standards and producing your calibration results using the international standards units. When using international standards, the calibration is normally performed by specialized engineers who are competent to perform such tasks.
How to Know If the Calibration Process is Completely Safe
- When performing your calibration process, ensure that you perform the measurements using the thermal characteristic of your thermal cyclers, such as the cool heat speed, retention time, excess, or reach uniformity and accuracy.
- Carry out a dynamic calibration process instead of a static calibration.
- When carrying out the calibration, endure that you have the necessary expertise and experience. Always ensure that you conduct your calibration exercises in controlled environmental conditions.
- Calibrate your thermal cycler using different channels that will enable you to eliminate the time effect between wells.
Calibrating your thermal cycler is essential for improving the accuracy of your tests, improving your PCR yields, controlling your reagents, and conforming to the regulatory requirements. Calibration of a thermal cycler is a sensitive exercise that a specialized and competent engineer must carry out. At Biotechnical Services, Inc., we have experienced personnel to calibrate your thermal cycler according to the required international standards. We provide accredited calibration and validation testing for all types of thermal cyclers. Our laboratory staff uses stringent practices that comply with the accredited standards. If you are in Los Angeles or any part of California, contact us today for the best thermal cycler lab equipment calibration services.