4BB™ SunScript® One Step RT-qPCR Kit is an easy and reliable system designed for fast RT-qPCR (real time) reactions based on detection of double stranded DNA binding dyes such as SYBR Green.

This RT-qPCR Kit allows for elevated temperature (60°C–75°C) when performing the reverse transcription reaction. This enables a higher specificity of the reaction and an increased likelihood in obtaining reaction products from difficult RNAs with a high degree of secondary structure or high Guanine and Cytosine (GC) content. Moreover, the excellent kinetic performance of the SunScript® Reverse Transcriptase allows for speed optimization of the one step RT-qPCR protocol, resulting in reaction times as low as five minutes for complete cDNA synthesis.

The 4BB™ SunScript® kit contains all the components needed to perform both reverse transcription and qPCR amplification (including SYBR Green I). By using gene specific primers, this one step reaction minimizes contamination and allows greater reproducibility.

Features and Benefits:

• RT step completed in as little as five minutes

• RT reaction temperatures of at least 75°C

• Outperforms one step RT-qPCR competitors

• Easy detection of low abundance gene products

• Broad dynamic range of quantification

• Accurate quantification for high quality standard curves

• Compatible with all qPCR machines (Sybr Green quantification).

Technical Specifications:
The system uses an enzyme master mix containing:

• 4BB™ SunScript® Reverse Transcriptase RNaseH+ a high quality HotStart Taq Polymerase,

• RNase inhibitor

• 2X buffer containing dNTPs, salts, stabilizers and the double stranded DNA binding dye.

4BB™ SunScript® Reverse Transcriptase RNaseH+ is an engineered version of the well characterized HIV-1 RT with increased thermostability. This enables to perform the reverse transcription step at temperatures up to 75°C if needed (the recommended temperature for the RT reaction is 60°C for most samples depending on the melting temperature of the primers). These features make this kit the best choice for amplifying a broad range of mRNAs, also with complicated secondary structures or high GC content.

1. The reverse transcription reaction can be effectively performed while Taq Polymerase remains inactive during this step due to blocking antibodies

2. The inactivation / activation step at 95°C provides an automatic hot start for the Taq Polymerase to amplify the reverse transcribed cDNA

3. Intercalated SYBR Green I can be detected using common SYBR Green I settings (excitation wavelength ~450nm, emission wavelength ~520nm).

The reaction mixes contain proprietary formulations that have been optimized for both the reverse transcription and the qPCR reaction, including dNTPs, MgCl2 and stabilizers resulting in a final concentration of 250nM each dNTP and 3.5mM MgCl2 in the final reaction.

The final recommended reaction volume is 20µl, suitable for all plate based qPCR instruments (e.g. Bio-Rad CFX series Real-Time PCR Detection Systems, Roche LightCycler™ 480, etc.).

Shipping and Storage:
This product is shipped on dry ice. Upon receipt, it should be stored immediately at -20°C in a non frost free (constant temperature) freezer. If stored correctly, the product can be kept for at least three months after shipping without displaying any reduction in performance. For longer periods of time, store the kit at -80°C. Avoid frequent freeze thaw cycles. Avoid long exposure of kit contents to light.

Quality Control:
Each batch of 4BB™ SunScript® One-Step RT-qPCR Kit and individual components are tested against predetermined specifications to ensure consistent product quality.

Intended Use:
4BB™ SunScript® One-Step RT-qPCR Kit is intended for molecular biology use only and in vitro use only. This product is not intended for diagnosis, prevention or treatment of a disease in human beings or animals.

Safety Information:
Safety data sheets are available for download. This material may contain substances or activities that are harmful to human health. It should not be ingested, inhaled, or brought into contact with skin, and handled with appropriate care in accordance with the principles of good laboratory practices.

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