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 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.
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.