FastGene® Scriptase II cDNA Kit (100 rxns) contains FastGene Scriptase II, enzyme buffer, dNTPs, Oligo dTs, random hexamers and RNase inhibitor
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.
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.
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.
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.
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.
We recommend to use 15 pmol – 20 pmol for gene specific primer
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.
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.