SYBR® Green is a minor groove binding dye that binds double-stranded (ds) DNA but not single stranded DNA. A signal from SYBR® Green will be detectable only after significant amplification (>15 cycles for most cDNA). Since SYBR® Green binds to dsDNA, all dsDNA present in the reactions (e.g. primer dimers or non-specific amplification products) will also be detected. Primers should always be validated to insure that a single amplicon is generated by the PCR. Reactions may be run on agarose gels to ensure that only one product is present. The thermal denaturation protocol on the Applied Biosystems and most other real-time PCR instruments should always be run following SYBR® Green reactions. This heats the products after the final reaction from 60°C to 90°C and will produce a characteristic peak at the Tm of each amplicon. If only one amplicon is present, then only one peak will be present. If primer dimers are present along with the product then a second peak (usually with reduced Tm) will be seen.
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