EvaGreen qPCR Mastermix-iCycler 4 x 1.25ml

Description	Guaranteed high-performance real-time PCR using abm’s EvaGreen 2X qPCR MasterMix. abm’s EvaGreen 2X qPCR MasterMix provides all ingredients necessary for quantitative PCR in a premixed and optimized format. Available with the option of ROX or fluorescein as the internal passive reference dye, abm’s EvaGreen 2X qPCR MasterMix offers unparalleled performance in sensitivity, signal-to-noise ratio, and complete elimination of primer dimers. Key Features • Enable streamlined protocol in a simple reaction set-up • Allow accurate quantification of a variety of gene targets • Reduce pipetting steps to minimize the risk of contamination • Compatible with most real-time PCR instruments Please refer to our qPCR MasterMix Selection Guide (found in the Protocol section below) for selecting the appropriate qPCR formulation applicable to your particular instrument model.
Application	• Gene-expression analysis • Gene knockdown validation • Microarray validation • High throughput applications • Virus detection and quantification
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Protocol
Storage	Stored at -20°C and protect from light.
Notes	1. Aliquot reagents to avoid contamination and to avoid repeated freeze-thaw cycles. 2. Primer concentration should not be high, a concentration of 100 nM to 300 nM of each primer usually gives the best results. 3. A very effective way to get tight Ct among replicates is to reduce pipetting error, this can be achieved by: preparing amplicon specific pre-mix, using repeating pipet, and keeping pipetting volume in manufacture suggested range. 4. For optimal results, it is recommended that the primers are 18-22 nucleotides in length with a Tm of 58°C-60°C and the size of target is about 100-250 bp. This product is distributed for laboratory research only. Caution: Not for diagnostic use.

FAQs

What is the difference between the fluorescent detection dye & reference dye?

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.

Do your qPCR products work for the FAST protocol?

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.

What is the difference between EvaGreen and SybrGreen? Which has better features?

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.

What are the main advantages for EvaGreen?

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.

Can I use the Mastermix R/LR/S/iC for Taqman probe?

No, the formulations are not compatible. Please refer to TaqProbe Mastermix series.

Do our qPCR mastermixes contain Uracil-DNA glycosylase?

No, they do not.

What the concentration of MgCl2 is in the EvaGreen Mastermixes?

The concentration in 2x Mastermix of Mg is 6mM. Thus the final concentration in qPCR reaction is 3mM.

The protocol of abm’s qPCR Mastermixes states a 2 steps thermal cycle instead of a normal 3 steps program, would it be a mistake in the protocol?

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.

Usually SYBR dyes doesn't require calibration; does EvaGreen?

EvaGreen does not require calibration.

Are EvaGreen Mastermixes suitable for experiments being setup under normal lighting?

EvaGreen Mastermixes are very stable and they are definitely suitable for setting up experiments under normal lighting.

I noticed that there are clear crystals in the product solution. Is it still functional?

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.

Is it correct that the Tm of the primers should be around the same as the annealing temp (~60C)?

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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