PCR System Kit

Expand High Fidelity PCR System Kit (Roche Molecular Biochemicals). 

Generate amplicons from the pOK-target locus plasmid with the inactivation or reconstitution endonuclease-tagged primer sets using the Expand Long Template PCR System Kit (Roche Diagnostics) per the manufacturer s instructions. 

On the basis of the size of the amplicon, two different PCR (20) systems were utilized for the construction of plasmids. Amplicons under 1000 bp were generated using a GeneAmp Core Kit (Roche Molecular Systems), and amplicons over 1000 bp were produced using an Expand Long Template PCR System Kit (Roche Diagnostics), per the manufacturer s instructions. 

A protocol combining commercial kits for RNA extraction (Amplicor HIV Monitor test, Roche, 073 0246) and one-tube RT-PCR (Titan One-Tube RT PCR system, Boehringer Mannheim, 1 855 476) was established. The procedure described herein is less sensitive than the one described in Subheadings 3.1.1.-3.1.3., because positive amplification results were obtained only starting from 3000 copies/mL. The following protocol was used ... 

InsTAclone PCR Cloning Kit (Fermentas Life Sciences K 1214) The kit contains a TA cloning vector (pTZ57R/T), 5x ligation buffer, T4 DNA ligase 5 U/gL, controls and Transform Aid bacterial transformation system with C medium and T-solution A and B. 

GeneAmp PCR Core Kit (Roche Molecular Systems, Branchburg, NJ). 

PCR products are labelled with the Universal Linkage System (ULS ) [3] using the Fluorescein ULS labelling kit from Fermentas Life Sciences. The procedure is as follows ... 

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. 

The next major change in the analysis of the DNA for paternity (and forensic) analysis incorporated the PCR amplification of microsatellites instead of minisatellites. Microsatellites are also formed by tandem repeats but consist of two to five nucleotides per repeat units. This means that the amplification requires less DNA (less than 1 ng) and the quality of the material may be less than ideal. This capability permits the analysis of some degraded DNAs. The potential number of loci is very large and the process is rapid it may be completed in a day or two. This system also has the benefit of lending itself to multiplexing and automation. In addition, several kits are available, and for some multiplexes inexpensive silver stain materials may be employed without expensive equipment. 

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