Thermal cycler

Even inside the controlled conditions of a research laboratory, analyzing clean and standardized test samples PCR procedures requires careful quality control, taking into consideration differences in sample preparation, variation in pipetting, differences in reaction tube thickness, poor calibration or instability of the thermal cycler, and reagent quality. 

DNA The template DNA is isolated from cells by some sort of extraction procedure. This is usually the last thing added to the reaction before the tube is placed in the thermal cycler. 

Once the reaction tube has been placed in the thermal cycler, there are normally three... 

Polymerase chain reaction (PCR) is one of the most important techniques for rapid bacterial identification. It consists of repeated cycles of enzymatic reactions in a thermal cycler (PCR machine) that copies DNA strands many times. The DNA amplified in one PCR cycle is used as a template for the next cycle. This results in an exponential increase of the desired target... 

The instrumentation for real-time PCR includes a thermal cycler with a computer, a spectrophotometer for fluorescence detection, and software for acquisition and analysis of data.105,109... 

MacPherson,J.M., Eckstein, P.E., Scoles, G.J. and Gajadar, A.A. (1993) Variability of the random amplified polymorphic DNA assay among thermal cyclers, and effects of primer and DNA concentration. Molecular and Cellular Probes 7, 293-299. 

FIGURE 11.8 Thermal cycler for PCR. Source http //www.intlmiss.com/... 

Since rolling circle amplification takes place at a constant temperature, there is no need for the target amplification process to take place in a thermal cycler, which is required to regulate the temperature for different parts of the reaction. The type of DNA polymerase to be used in RCA is not limited to thermostable enzymes, like the PCR-based diagnostics. On the other hand, the RCA method requires the environment to be free of contaminations as the RCA arrays are highly sensitive. Wiltshire [22]... 

Exogenous sources such as a person s hair or skin, doorknobs, laboratory benches, dust, reagents, thermal cyclers, and pipet tips are some of the common sources of DNA contamination. Ideally, a laminar air flow bench with filtered air provides a clean, dust-free environment. Sample preparation should be done in a separate room or area. The addition of sample to the PCR reaction mixture in the... 

Early PCR experiments were performed by manual or robotic transfer of reaction vials between water baths or heating blocks. These have been superseded by several generations of programmable thermal cyclers. Successful PCR amplifies a single or a few copies of a target sequence of DNA by many orders of magnitude. The process is described schematically in Figure 2.9. 

In situ PCR machine (Thermal Cycler PTC-100 M. J. Research, Watertown, MA). 

Place these slides in a Thermal Cycler and heat them for 10 min at 95°C. 

Leave sealed slides on the bench top for 5-7 min to dry the rubber cement. Insert slides into a Thermal Cycler and run a program for RT reaction. 

At the end of the incubation, quickly remove all slides from the Thermal Cycler and place on ice for 1 min. 

Program the Thermal Cycler to keep the slides at 22-25°C at the end of the PCR cycles (if you plan to leave the slides overnight in the machine). 

Heat the slides at 92°C for 1-2 min either in a Thermal Cycler or on a heat block in order to immobilize the signal. 

Place the covered slides on a PCR thermal cycler with slide blocks or a hybridization instrument. 

There is usually no calibration required. Thermal cyclers should be checked and calibrated by the manufacturer at least once every year. 

A thermal cycler (see 8.2.3.2 Polymerase Chain Reaction) is required for the cycle sequencing reaction. For fluorescent cycle sequencing we recommend instruments from Applied Biosystems (e.g. the 16-capillary ABI PRISM 3100 Genetic Analyser) other instruments are available from GC Healthcare and Beckman. For detailed instructions, refer to the respective user s manual or chemistry guide. 

For mapping single-copy genes, the incubation times in the thermal cycler should be longer. The anneal log temperature should be between 54-60°C. The incubation time may be extended to 10 min. The primer extension step at 72°C should be extended to 30 min... 

Digoxigenin-labeled chromosome 10 a-satellite probe (DIOZI Oncor, Gaithersburg, MD) is used at a final concentration of 5 pl/100 pi in hybridization buffer (50% formamide, 10% dextran sulfate, 0.004% Tween 20, and standard saline citrate (SSC) [in 1.5 strength]). A 10-pl volume of the probe is placed on an 18 x 18-mm coverslip, which is placed onto the slide, sealed with special Vulcanizing Fluid, and placed on a flat-bed thermal cycler. Slides are incubated for 3 min at 94°C and placed in a humidified box for 16 hr at 37°C. 

Zhou et al. [175] described the determination of severe acute respiratory syndrome (SARS) coronavirus by a microfluidic chip system. The unit included an LIF microfluidic chip analyzer, a glass microchip for both PCR and capillary electrophoresis, a chip thermal cycler based on dual Peltier thermoelectric elements, a reverse transcription-polymerase chain reaction (RT-PCR) SARS diagnostic kit, and a DNA electrophoretic sizing kit. According to the authors, the system allowed efficient DNA amplification of the SARS coronavirus followed by electrophoretic sizing and detection on the same chip. 

Integration of sample preparation and analysis [46] is one of the prime objectives of /i-TAS. PCR on a chip is one of the earliest applications of sample preparation. It has been carried out in the sample reservoir of the electrophoretic chip shown in Figure 8.22a. The nucleotides, primers, and other chemicals are added into the sample reservoir, and the entire device is introduced into a conventional PCR thermal cycler. The PCR products from the sample reservoir are then injected into the separation channel and analyzed. A more complex chip with multiple PCR chambers is shown in Figure 8.22 b. 

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