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The detection dye (EvaGreen) is DNA binding dye only emits fluorescent at certain wavelength after binding to dsDNA; it reports the presence and helps quantifying of the amplified DNA.
Reference dye (ROX for example) is a fluorescent dye which emits at a different wavelength (ie different channel) from the EvaGreen's emission channel (no detection interference);
and absorbs at a wavelength away from the emission channel of EvaGreen (no signal quenching).
Although reference dye can normalize non-PCR-related fluctuations in fluorescence and provides a stable baseline for multiplex quantitative PCR and RT-PCR,
the main purpose of reference dye to to normalizes/correct the "well-to-well" variation of the instruments such as the difference in light paths between wells.
All of our qPCR lines (except for the Express line) can handle the FAST protocol. The Express qPCR line has an unique protocol that is even faster than the FAST protocol.
EvaGreen is new generation of SybrGreen which gives a stronger signal when bound to dsDNA compared with SYBRGreen, it also has reduced toxicity. EvaGreen provides much higher sensitivity and better low-copy detection results.
EvaGreen can provide high sensitivity in a ready-to-use format (other companies may require a separate reference dye) for any qPCR instrument on the market.
No, the formulations are not compatible. Please refer to TaqProbe Mastermix series.
No, they do not.
The concentration in 2x Mastermix of Mg is 6mM. Thus the final concentration in qPCR reaction is 3mM.
It is not a mistake in the protocol, a 2 steps thermal cycle program is very common in qPCR and the results is equivalent to those obtained from a conventional 3 steps thermal cycle qPCR experiment but only using a fraction of the time. abm’s qPCR matermixes is fully compatible with both quick 2 steps thermal cycle and conventional 3 steps thermal cycle programs.
EvaGreen does not require calibration.
EvaGreen Mastermixes are very stable and they are definitely suitable for setting up experiments under normal lighting.
The qPCR mastermix should still be functional. The crystals are from our proprietary buffering salt, which will be dissolved eventually when you warm the solution up to room temperature or even 37C. All our qPCR mastermixes are very stable and can withstand this kind of warming cycle. Once the salt is fully dissolved, the vial can be kept at 4C for 2-3 months.
Yes, in qPCR having a Tm close to the annealing temperature will allow for much more specific priming. In turn, this may lead to a reduced yield when compared with PCR, however you should find the amplified end product is much more accurate and specific to the intended target.
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