TOPscript™ III Reverse Transcriptase (High fidelity) is a blend of a recombinant reverse transcriptase and a proofreading enzyme. The synergistic effect of both enzymes generate 7-fold higher fidelity compared to other commonly used reverse transcriptases. Due to the higher thermostability of reverse transcriptase and the specially optimized buffer system, reverse transcription reaction is possible at temperatures up to +55℃. TOPscript™ III Reverse Transcriptase (High fidelity) does not degrade RNA strands from RNA-DNA hybrids and therefore is capable of high yield of synthesizing cDNA. The optimum temperature of TOPscript™ III Reverse Transcriptase (High fidelity) is 50℃. TOPscript™ III Reverse Transcriptase (High fidelity) does not show inhibitory effect on one-step RT-PCR reaction.
- Thermostable and RNase H variant of M-MLV RTase
- Synthesize cDNA at 42℃~55℃.
- Especially the highest activity at 50℃
- Generate full-length transcripts up to 14 kb with the oligo(dT) primers.
- Increase accuracy during reverse transcription reactions.
- Efficiently synthesize cDNA by inhibiting the formation of secondary structure of RNA.
- Cloning genes of interest
- Sequencing transcriptomes
- Generating cDNA libraries with large and full-length inserts
- Generating gene expression analysis, like RNA splicing analysis.
One unit is the amount of enzyme required to incorporate 1 nmol dTTP into acidinsoluble materials using 0.4 mM poly(rA)-oligo(dT) as substrate at 37℃ in 10 min.
- 10X TOPscript™ RT Buffer
- dNTP Mixture (2 mM each)
- Sterile water (RNase free)
20 mM Tris-Cl (pH 7.5), 100 mM NaCl, 1 mM DTT, 0.1 mM EDTA, 0.01% NP40, 50% glycerol
10X TOPscript™ RT Buffer
500 mM Tris-HCl (pH 8.3), 30 mM MgCl, 100 mM DTT, 750 mM KCl
TOPscript™ Reverse Transcriptase performs optionally over the full range of 42℃-60℃. Typically, 50℃ is a good starting point. For RNAs containing secondary structure and other challenging targets, a synthesis temperature of 60℃ may be used without loss of performance.