DNA thermal cycler

Sterile thin-walled microcentrifuge tubes for the reaction mixtures of appropriate size to be placed in the DNA thermal cycler. 

Millipore Ultrafree MC filters (0.45 m. Cat. No. UFC3 OHVOO) were from Millipore Corporation. X-ray film (Kodak X-Omat-AR) was obtained from the local Kodak supplier. The DNA thermal cycler and Taq polymerase Ampli Taq (Cat. No. 3128) were obtained from Perkin-Elmer-Cetus. All electrophoresis apparatus were conventional homemade (workshop of Ciba-Geigy AG) equipment. 

Overlay vrith 100 pi of mineral ofr and place the tubes in a DNA thermal cycler preset at 94 °C (Hot Start). 

The use of mineral oil can be avoided when using a DNA thermal cycler equipped with a heated lid. 

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. 

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... 

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. 

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. 

A thermal cycler (see 8.2.3.2 Polymerase Chain Reaction) is required for the cycle sequencing reaction. MRC Holland offers kits for different sequencing instruments. For more detailed instructions refer to the appropriate instrument user s manual. MLPA fragment analysis requires a fluorescent DNA analysis system. 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. 

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. 

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