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 cDNA 5x ReadyMix OdT comes with all necessary ingredients to perform a revers transcription:
- FastGene® Scriptase II
- 5x FastGene® Scriptase II buffer
- dNTP Mixture
- RNase Inhibitor
- Random Hexamer
- Oligo dTs
- Helper Protein
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.
Can I use LS65 with RNA in a solution which contains DNaseI inactivated by EDTA and heat.
Heat and EDTA does not inactivate DNaseI by 100%. To avoid genomic DNA we recommend to use RNA Premium Kit (FG-81#). In this mentioned setting, the EDTA will block the activity of Scriptase II, because the EDTA chelates the Ions. To support the Scriptase II we recommend to add MgCl2 to compensate the EDTA effect.
EDTA chelates 2 molecules of Mg2+. Just calculate the amount of MgCl2 which has to be added to the reaction solution.
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