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99 (10)

of MTP slide of MTP slide

500 bp 250 bp

Fig. 2 Coamplification of internal PCR control. All PCR reactions contain primers for the specific pathogen (here, a 236-bp segment of hepatitis B virus, or HBV, is amplified) and for the internal PCR control template IC (here leading to a 400-bp amplicon). The IC sequence and primers are unrelated to the target pathogen and all PCR reactions are spiked with a defined amount of IC. In the absence of the pathogen (negative controls and negative samples), amplification of IC indicates proper PCR conditions and the absence of PCR inhibitors. These inhibitors frequently occur in biological samples and have to be removed during isolation of the DNA, prior to PCR analysis. Theoretical dilution down to one HBV copy per reaction frequently leads to no copy and thus to negative results. At low HBV titers (up to 100 copies per PCR reaction), amplicons from IC and HBV are observed. At higher HBV titers (1000 copies and more), only the HBV-specific amplicon is generated due to competition for reaction compounds. (View this art in color at www.dekker.com.)

number of input target copies (T): P=TEn (E=PCR cycle efficiency, ideally 2.0; n=number of PCR cycles).[8] However, this calculation is based on a constant value for E, and this requirement is met only for a limited number of cycles, the logarithmic or exponential phase. With increasing product concentration and stepwise consumption of primers and other reaction components, E will decrease and, finally, a plateau phase is reached with E =1 (Fig. 4). In consequence, product quantities reflect the initial target amounts only until certain cycle numbers are reached, whereas this relation is lost at higher cycle numbers (Fig. 5). In practice, unknown target amounts require the comparative quantitative analysis of PCR products after the completion of several different PCR cycle numbers. Again, real-time PCR techniques are preferred in routine applications. However, the previously mentioned sequence-specific detection is not mandatory and a simplified and much cheaper detection of amplicons by means of intercalating dyes such as ethidium bromide or SYBR Green is sufficient (see ''Real-Time PCR Technology'').

Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

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