FastGene Scriptase II cDNA 5x ReadyMix OdT

Details

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.

Composition

Ready-to-use

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

Protocol

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

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.

Documents

Manual

LS65_Scriptase II – Ready Mix_Manual

Technical Note

Very fast reverse transcription reactions (5min)

Application Notes

Cloning of isoflavone biosynthesis related enzyme using RNA extracted from soybene

Comparative study of reverse transcriptase reaction using RNA extracted from zebrafish fertilized eggs

Reverse transcriptase reaction for quantitative expression analysis using RNA extracted from mesenteric adipose rat tissue

Comparative study of RT-reaction by using RNA extracted from mouse lymph node

Comparative study of reverse transcriptase reaction using RNA extracted from peritoneal cells of sterile peritonitis model mouse

Oligo dT verification of FastGene® ScriptaseⅡ cDNA 5x ReadyMix OdT

MSDS

LS65_FG Scriptase II cDNA 5x Ready Mix OdT_MSDS

FAQ

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.

Product inquiry

Do you have any questions about the product? Please contact us!