FastGene Scriptase II cDNA Kit

Ligation of DNA strands

FastGene® Scriptase II cDNA Kit (100 rxns) contains FastGene Scriptase II, enzyme buffer, dNTPs, Oligo dTs, random hexamers and RNase inhibitor

Price on request


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Cat. No. LS63

Details

Engineered enzyme

Engineered reverse transcriptases allowed the synthesis of cDNA from very low amounts of RNA. Mutations are inserted into the RNase H domain of the MuLV‘s reverse transcriptase. Therefore, by not degrading the RNA during the first-strand synthesis, a higher yield of full-length cDNA is obtained. Additionally, a higher thermal stability increases the robustness of the enzyme. The FastGene® Scriptase II is exactly one of those engineered enzymes. With its mutation in the RNase H domain and higher thermal stability, it is the optimal choice for more complex applications, such as RT-qPCR and NGS.

Applications
  • Quantification of Gene Expression
  • RT-qPCR
  • Next Generation Sequencing
  • Low RNA concentration
  • Difficult templates
Lower RNase H activity for longer cDNA

The FastGene® Scriptase II has a modified RNase H domain. The RNA is therefore not degraded and serves as a template for longer cDNAs, resulting in fragment size of up to 12 kBp.

Engineered enzymes – optimized for qPCR

The FastGene® Scriptase II delivers superior cDNA templates for downstream applications, e.g. qPCR and NGS. The resulting full-length cDNA gives a complete picture of the gene and is able to show modification, e.g. splicing variants.

Comparison of different PCR results
Multiplex PCR

Fig. 1: Comparison of multiplex PCR using cDNA produced by Competitor I‘s SS-II enzyme and FastGene® Scriptase II at 42 °C and 50 °C.

qPCR

Fig. 2: Comparison of qPCR results using primers for GAPDH and cDNA produced by using different RNA starting concentration by Competitor I‘s SS-II enzyme   and FastGene® Scriptase II at 42 °C.

Fig. 3: Comparison of qPCR results using primers for YWHAZ and cDNA produced by using different RNA starting concentration by Competitor I‘s SS-II enzyme and FastGene® Scriptase II at 42 °C.

Documents

LS63_FG-Scriptase-II-cDNA-Kit_TDS_vers.2020
Application Note
Technical Note
Application Note
Application Note
Application Note
Application Note
MSDS

FAQ

Which concentration of gene specific primer for LS63 is recommended?

We recommend to use 15 pmol – 20 pmol for gene specific primer


What is the purpose of the step 42°C for 2 minutes and will the yield decrease significantly if I skip this step? If I use oligo dT primer can I skip this step?

The purpose of this step is the annealing of primer. For oligo dT it is basically recommended to have an annealing step at 42°C for 2 minutes, because it has low Tm.

The yield is not influencend if the amount of template is sufficient and the primers work well (good designed). In this case you can skip the step. But if the amount of template is small and the primer are not optimized we expect that there will be an effect to the yield.

In the protocol the annealing step for oligo dT is a standard recommendation. For certain targets, the condition of 40°C for 2 minutes or 37°C for 2 min and so on, could be better for real time experiments than 42°C. This means it is possible that you need to optimize empirically the reaction temperature, time and step for your target.


Can I use the Scriptase II cDNA Kit with a high amount of RNA (2.5 µg per 20 µl)?

We recommend a max of 1 µg. Reason: the highly expressed genes will over proportionally be represented in such high concentration. Meaning the chances of finding a high-concentrated mRNA is much higher than a low concentrated one. If you stick to 1 µg, the amount of enzyme to mRNA will be much higher, guaranteeing the complete RT of all mRNA.


Is this Enzyme influenced by DEPC?

If DEPC is not completely removed, it can inhibit all enzymatic reactions including Scriptase II.

4 reviews for FastGene Scriptase II cDNA Kit

  1. Haruko Hayasaka Immunohole Functional Laboratory, Department of Bioscience and Biotechnology, Kinki University

    I especially like that the Scriptase II lead to stable results. As a result of performing RT-PCR using tumor derived RNA, we were able to detect the expression of genes whose amplification was unstable with other RT reagents. The Amplification of full-length cDNA has also been confirmed. I would love to also try the 5x Ready Mix.

  2. Ryo Mameda, Department of Biomolecular, Graduate School of Engineering, Tohoku University, Japan

    PCR amplified DNA fragement was cloned into a vector and the sequence was confirmed. As a result, there was no artificial mutation such as changes of single nucleotides.

    Also, the manual of your product (FastGene® Scriptase II cDNA Synthesis Kit) was very easy to understand and I felt no stress on the operation. I definitely want to use other products from NIPPON Genetics.

  3. Dr. Catherine Moermans, pneumology department, CHU-ULiège, Liège, Belgium

    “The FastGene® scriptase II cdna synthesis kit appeared to be a reliable method to perform RT-PCR experiments using induced sputum samples which is a difficult specimen matrix. Indeed, it allowed to obtain a good reproducibility and good amplification curve profils. Furthermore, the cost of this product is really cheap compared to what we use to buy.”

    Translation by Nippon Genetics:

    Das FastGene® Scriptase II cDNA Kit zeichnet sich als zuverlässige Methode aus um RT-PCR Experimente mit Proben eines induzierten Suptums durchzuführen. Obwohl dies eine schwere Probenmatrix ist, konnte eine gute Reproduzierbarkeit und ein gutes Amplifikationskurvenprofil gewonnen werden. Zusätzlich sind die Kosten verglichen zu dem zuvor verwendetem Produkt sehr preisgünstig.

  4. Nathalie Renotte, Cellular and Molecular Epigenetics, GIGA Cancer, Liège, Belgium

    “We have tested the RT kit FastGene Scriptase II in comparison with other kits, like Protoscript II (NEB) and Superscipt II (Invitrogen) and we have noted that the results are similar. Additionally, we can make more “runs” for a better price!”

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