PrimeTime¢ç qPCR 5' Nuclease AssaysPrimer, Probe mix
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PrimeTime¢ç qPCR 5' Nuclease Assays¿¡ °¡´ÉÇÑ Reporter ¿Í Quencher Á¶ÇÕ
Pre-designed PrimeTime¢ç qPCR Assay* 100 % °á°ú º¸Àå (positive¿Í negative control µ¥ÀÌÅÍ Á¦°ø ½Ã)
* Sequence Á¦°ø (Á¦Ç°°ú µ¿ºÀ)
* Human, mouse, rat ¼¼ °¡Áö species¿¡¼¸¸ µðÀÚÀÎ Á¦°ø
Pre-designed PrimeTime¢ç qPCR Assay´Â¾Æ·¡ ¸µÅ©¿¡¼ µðÀÚÀÎÀ» Á÷Á¢ ÇÒ ¼ö ÀÖ½À´Ï´Ù.http://sg.idtdna.com/order/predesignedassay.aspx?source=scitoolsOverview of 5¡Ç Nuclease AssaysStep 1—The primers and probe hybridize in a sequence-dependent manner to the complementary DNA strand. Because the probe is intact, the fluorophore and quencher are in close proximity and the quencher absorbs fluorescence emitted by the fluorophore.
Step 2—The polymerase extends from the primers and begins DNA synthesis.
Step 3—The polymerase reaches the probe and the exonuclease activity of the polymerase cleaves the hybridized probe. As a result of cleavage, the fluorophore is separated from the quencher and fluoresces.
Step 4—The fluorescence is detected by the real time instrument.
These steps are repeated for each PCR cycle and allow detection of specific products. With intercalating dyes, such as SYBR¢ç Green I, primer-dimers and non-specific products will also contribute to fluorescence. In contrast, the 5¡¯ Nuclease assay is specific and fluorescence will only be detected for the DNA sequence to which the probe and primers hybridize.
Selecting the Correct Reporter Dye and QuencherReporter Dyes
The correct reporter dye will depend on the type of instrument you are using and the compatibility of the dye with the instrument. Please see the Instrument Compatibility Table for a list of reporter dyes compatible with common instrumentation.
For multiplexing applications, it is recommended that reporters dyes with the least amount of spectral overlap be selected. For a complete list of IDT¡¯s dyes and quenchers please see the Dye and Quencher Wavelength Figure and the Instrument Compatibility Table
Quenchers
Traditional dark quenchers absorb broadly and do not emit light which allows for the use of multiple reporter dyes with a given quencher. This characteristic allows for expanded options for multiplex assays. Dark quenchers simplify detection which makes them compatible with a broad range of image analysis instruments.
IDT has developed an internal ZEN quencher that enables to production of Double-Quenched Probes which have less background and more signal. The Double-Quenched Probes contain a 5¡¯ FAM fluorophore, a 3¡¯ IBFQ quencher, and an internal ZEN quencher. These Double-Quenched Probes are an improvement over traditional dual-labeled probes and have consistently low background, reduced Cq values, improved precision, and enable the use of longer probes for design in AT-rich regions. For more information download the ZEN Double-Quenched Probe Overview.
IDT supplies commonly used dark quenchers as well as the proprietary dark quenchers, Iowa Black FQ, Iowa Black RQ, and the newly developed internal ZEN quencher. TAMRA is also a quencher option for a FAM reporter dye. Related Information
primetime-qpcr-application-guide-3rd-ed-.pdf (8,682,050kb) zenoverview.pdf (1,202,074kb) qpcr-resuspension.pdf (159,593kb) |
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