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Real-Time Quantification of Genomic DNA Using DyNAzyme II DNA Polymerase and SYBR Green I Dye

e valuable when analyzing small populations of cells or trace amounts of DNA.

Protocol Adaptable for a Wide Variety of Applications
The protocol presented here can be applied to a wide variety of PCR applications by following several guidelines to modify the protocol for work with SYBR Green I. Because SYBR Green I stabilizes dsDNA, the denaturing temperature of the cycling protocol should be raised approximately 2C in order to ensure complete denaturation of the duplex. In addition, it is recommended that the annealing temperature of PCR primers be optimized for the SGI reaction conditions. This is accomplished by running test reactions with an annealing temperature gradient. The temperature gradient can be programmed into the cycling protocol through the Opticon Monitor software. Melting curves and agarose gel data can be used to evaluate yield and specificity for the target amplicon.

DMSO for the Most Challenging Templates
A wide variety of samples can be analyzed with the protocol described here. The human genomic DNA amplified with -actin primers represents an optimal sample in terms of purity and ease of amplification. However, amplification of challenging templates may benefit from modification of reaction conditions, such as the addition of DMSO. The destabilizing effect of DMSO on double-stranded DNA reduces the denaturation temperature of the amplicon thereby facilitating the amplification of templates with strong secondary structure. Success has been experienced with the addition of DMSO in concentrations of up to 5% (vol/vol). Caution must be used when adding DMSO to reactions, as the destabilizing effect on dsDNA will result in a decrease of bound SGI, and red
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