Realtime qPCR with RTGreen (Eva Green )

Realtime qPCR with RTGreen(Eva Green)

Cat.:M00480

Size:50uL

Concentration: 20× (25 μM) in water

Color and Form: Light orange solution

Storage:-20℃,keep from light,1 year

Product Description

RTGreen(Eva Green) is a very sensitive dye for the detection of double stranded DNA (dsDNA). The dye is a green fluorescent nucleic acid dye with features that make it useful for non-specific detection of amplification in realtime qPCR experiments.

Compared with the widely used SYBR Green I, RTGreen dye is generally less inhibitory toward PCR and less likely to cause nonspecific amplification , RTGreen dye can be used at a much higher dye concentration than SYBR Green I, resulting in more robust PCR signal.

The PCR reaction can be monitored using our existing optical setting for SYBR Green I or FAM on any commercial real-time PCR cycler. The qPCR protocol provided below is for PCR using regular non-hot-start Taq. Use of a hot-start Taq may require some adjustment of PCR buffer composition in terms of ionic strength and pH to best take the advantage of RTGreen dye. The water soluble solvent such as DMSO or glycerol are frequently added to stabilize master mixes. These components and pH may need to be optimized depending on the enzyme used.

Protocol

Calculate the volumes of reagents required for the reaction.

1. On ice, prepare a 1× master mix containing no DNA, by mixing the components in the following order: water, Taq polymerase buffer, dNTPs, MgCl₂, RTGreen, Taq polymerase, and primers.
2. Transfer master mix to tubes or plates. Add DNA (50 ng per reaction).
3. Proceed with amplification according to your instrument manufacturer. Perform real-time PCR on a thermocycling fluorometer and record the fluorescence signal at the annealing or extension step.

Notes:

1. Always use positive and negative controls when doing qPCR experiments.
2. The temperature program for the qPCR amplification does not differ from standard PCR program for the given template and primers.
3. For the detection, FAM or FAM/SYBR channel should be used.
4. When using ABI Sequence Detection Systems, make sure to select NONE for the passive reference under the tab WELL INSPECTOR.
5. BSA may be required if the reaction is run on a Roche LightCycler. A final BSA concentration of 0.5 mg/mL may be sufficient. With SYBR Green, addition of a protein such as BSA results in a fluorescence increase, which provides a background signal that triggers the start of a LightCycler. Because RTGreen dye is less sensitive to proteins, you may need to adjust the instrument setting (for background fluorescence) so that the instrument will start.