UltraScript Reverse Transcriptase is a robust and thermostable modified MMLV reverse transcriptase engineered to enhance cDNA synthesis speed and yield with accurate transcript representation.
The enzyme allows highly sensitive reverse transcription of viral RNA sequences, making it an ideal component of RT-qPCR-based SARS-CoV-2 detection tests.
- Recommended product for reverse transcription step of COVID-19 diagnostic tests
- Sensitive and accurate reverse transcription of viral RNA sequences, including SARS-CoV-2
- Thermostable reverse transcriptase 45°C to 55°C
- Advanced RNase inhibitor
- High cDNA yields from as little as 4pg total RNA
- Accurate reverse transcription of GC-rich templates
- Sensitive detection of low copy number transcripts
- Advanced buffer chemistry including Mg and dNTPs
UltraScript Reverse Transcriptase is a thermostable and extremely active modified MMLV reverse transcriptase (RTase). The reaction temperature can be increased up to 55°C providing higher specificity and efficient transcription of GC-rich RNA regions with a high secondary structure. The RTase is blended with an advanced RNase inhibitor preventing degradation of RNA by contaminating RNase. UltraScript Reverse Transcriptase is not inhibited by ribosomal and transfer RNAs, making total RNA an ideal substrate.
The enzyme is supplied with a 5x buffer containing Mg, dNTPs, stabilizers and enhancers, optimised to generate high yield cDNA from as little as 4pg RNA. As oligos are not included, UltraScript Reverse Transcriptase provides the convenience and flexibility for users to define their own priming strategy depending on the type of analysis needed. UltraScript Reverse Transcriptase gives exceptional performance with gene-specific primers, oligo(dT) and random hexamers to produce high quality cDNA ideal for a variety of downstream applications.
- Random hexamer, oligo(dT) and gene-specific primers
- cDNA synthesis for PCR analysis, cloning, library preparation and Next Generation Sequencing
- Low copy number transcripts
- Viral RNA targets
- miRNA targets
- Efficient synthesis from total RNA or poly(A)+ RNA
The kit should be stored between -30°C and -15°C. Avoid prolonged exposure to light. If stored correctly the kit will retain full activity for 12 months. The kit can be stored at 4°C for 1 month. The kit can go through 30 freeze/thaw cycles with no loss of activity.
Figure 1. Thermostable enzyme up to 55°C
The thermostability of UltraScript Reverse Transcriptase was measured using the Sypro Orange fluorescence assay. The protein is incubated with Sypro Orange dye and the temperature gradually increased. The fluorescence intensity increases
as the protein unfolds and the melting point
is the temperature where 50% of the protein is unfolded. The DSF curve shows UltraScript Reverse Transcriptase (purple) and wildtype MMLV RT (black) at 0.1mg/ml. This experiment shows that UltraScript Reverse Transcriptase unfolds at 54.9±0.3°C, which is 9.3°C higher than the wildtype enzyme, indicating it is more thermostable and more likely to remain active during the reaction.
Figure 2. Generation of high cDNA yields
cDNA was created from 100ng of total RNA from mouse liver using UltraScript Reverse Transcriptase (purple) and a competitor mix (blue). 5μM oligo-dT(18) and 5μM random hexamers were added as primers. The reaction was incubated for 10 minutes at 42°C. The resulting cDNA was quantified using qPCRBIO SyGreen Mix. PCR Biosystems UltraScript Reverse Transcriptase created >10x more cDNA than the competitor in the same amount of time.
|PB30.12-01||2 x 25μL 10’000 Units|
|PB30.12-04||2 x 100μL 40’000 Units|
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|UltraScript (200u/μL) with RNase inhibitor||
2 x 25μL: 10’000 Units
2 x 100μL: 40’000 Units
|5x UltraScript Buffer||
1 x 200μL: 10’000 Units
4 x 200μL: 40’000 Units