FastGene Scriptase II - Ready Mix (100 rxns)

Catalogue number: LS64

FastGene® Scriptase II cDNA Synthesis 5x Ready-Mix (100 rxns)

Ready-to-use with all necessary ingredients to perform a revers transcription.

Scriptase II cDNA Synthesis 5x Ready-Mix contain no Oligo dTs. For the version with Oligo dTs click here.

 

Applications

  • Quantification of Gene Expression
  • RT-qPCR
  • Next Generation Sequencing
  • Low RNA concentration
  • Difficult templates

Ready Mix

The FastGene® Scriptase II 5x ReadyMix is an engineered enzyme with a reduced RNase H activity and comes ready-to-use with all necessary ingredients to perform a revers transcription.

 

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.

 

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.

Ready-to-use

The FastGene® Scriptase II 5x ReadyMix comes with all necessary ingredients to perform a revers transcription:

 

  • FastGene® Scriptase II
  • 5x FastGene® Scriptase II buffer
  • dNTP Mixture
  • RNase Inhibitor
  • Random Hexamer
  • Helper Protein

1. Mix the ReadyMix with the RNA Template using the following setup:

5x FastGene® Scriptase II ReadyMix (LS64)4 µl
High quality RNA-template*x µl
Sterile Water up to 20 µl

*Concentration of template RNA can be up to 2.5 μg

 

2. Incubate at 25 °C for 10 minutes.

3. Incubate at 42 °C for 60 minutes*.

*Note: The recommended incubation time can be reduced to 5 minutes depending on the product abundance and size. Download here our Technical Note showing the performance at lower temperature (down to 5 min.).

 

4. Inactivate the FastGene Scriptase II by incubating at 85°C for 5 min.

5. Store the cDNA at -20 °C or use it for downstream application.

What is the recommended temperature for the Ready Mix?

The Scriptase II Ready Mix can only be used at 42°C, because there is a helper protein in this mix.


 

Can I use the Scriptase II Ready Mix for 15 min 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 that in 15 min the chances of finding a high-concentrated mRNA is much higher than a low concentrated one. If they stick to 1 µg, the amount of enzyme to mRNA will be much higher, guaranteeing the complete RT of all mRNA.

on 07/16/2019

Wir haben das Produkt ausprobiert und wir waren mehr als zufrieden damit! Wir haben verschiedene qPCRs durchgeführt mit zwei unterschiedlichen Kontrollen (GAPDH und Tubulin-b), sowohl für Zellinien, als auch für Patienten Proben (Biobank tiefgefrorenen Biopsiegewebe), und die Ergebnisse, waren ganz gut! Die Kontrollwerte sind stabil geblieben im Vergleich zu unserem alten cDNA Protokoll und sogar mit weniger RNA als früher. Anwendung und Protokoll fanden wir ebenso super easy!

Translation by Nippon Genetics:

We use the Sciptase II Ready Mix and we were more than happy with it! We performed different qPCRs with two different controls (GAPDH and tubulin-b), for both cell lines and patient samples (Biobank frozen biopsy tissue). The results were quite good! The control values were stable compared to our old cDNA protocol, even with less RNA than before. The application and the protocol were very easy!
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