NovaScript III is a premium ultrapure, highly sensitive RNase H minus Reverse Transcriptase offering higher & superior cDNA yields with distinct stability over a wide range of temperatures. |
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Super Agarose NS 3:1 is a molecular biology grade, standard melting temperature agarose that yields strong gels for fine resolution of small DNA, RNA and PCR products less than 1 Kb. Agarose 3:1 ensures of DNA fragments up to 1,000 bp fine resolution. This agarose can also resolve DNA fragments 10-1500 bp. Agarose 3:1 is designed for analytical electrophoresis. If you prefer using low melt temperature agarose we suggest AG-NSGTG which allows for enzyme cloning compatibility. |
Applications
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- NovaScript III is suitable for first strand cDNA synthesis in cDNA library construction.
- For the production of templates for RT-PCR analysis.
- NovaScript III can be used with total RNA, mRNA or in vitro transcribed RNA.
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DISCUSSIONS |
A successful reverse transcription reaction is dependent on many factors, including the reverse transcriptase used in the reaction. The enzyme should be able to generate high quality full-length cDNA from different types of templates and under various conditions. Commercially available reverse transcriptases include the Moloney Murine Leukaemia Virus (MMLV) reverse transcriptase and the Avian Myeloblastosis Virus (AMV) reverse transcriptase. These retroviral enzymes exhibit RNA-dependent DNA polymerase activity and RNA-degrading RNase H activity. Many variations of these enzymes are currently available in the market, including the wild type MMLV and AMV, which display full RNase H activity, and variations of MMLV which either lack the RNase H domain or contain its modified version, which greatly reduces its basal activity.
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NOVASCRIPT III RNase H minus Reverse Transcriptase
Description |
NOVASCRIPT III is a Moloney Murine Leukaemia Virus RNase H minus Reverse Transcriptase that exhibits high stability and is active at a wide range of temperatures. NovaScript III is an RNA and DNA-dependent DNA Polymerase requiring a DNA primer for initiation of elongation. Unlike wild-type enzyme, NovaScript III possesses RNase H minus activity which results in enhanced yields. NovaScript III is highly sensitive even when the amount of template is a limiting factor and is also suitable for real-time PCR experiments.
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NovaScript III produces high yield of cDNA, which shows excellent performance in realtime RT -PCR experiments
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Reverse transcription polymerase chain reaction (RT-PCR) is the most sensitive technique for mRNA detection and quantification currently available.Good quality cDNA is essential to this technique when high sensitivity is desired. The superior quality and performance of cDNA synthesized by NovaScript III is ratified vide end-point and real-time RTPCR assays.
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NovaScript III shows excellent performance with genespecific primers, oligo (dT)18 as well as random hexamers, and synthesizes cDNA with more splice variants--
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NovaScript III shows superior performance in reverse transcription of long templates
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For many applications full-length cDNA of long templates is required. To test the performance of NovaScript III for the synthesis of long transcripts a number of experiments were successfully carried out
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NovaScript III synthesizes high yield of cDNA over a wide range of temperatures
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Many RNA transcripts form stable secondary structures at lower temperatures, making them less suitable as templates for RT-PCR at those temperatures. Therefore, it is important that a reverse transcriptase is not only active at 37°C-42°C but also at higher temperatures without a loss of activity. The superior quality and performance of cDNA synthesized by NovaScript III is ratified at much higher temperatures. The Ct values for NovaScript III cDNA at higher temperatures is lower than the Ct values for the competing enzymes.
NovaScript III is suitable for first-strand cDNA synthesis, cDNA library construction and the production of templates for RT-PCR analysis of gene expression. NovaScript III can be used with total RNA, mRNA and in-vitro transcribed RNA and shows excellent performance with gene-specific primers, Oligo (dT) as well as random hexamers. It is highly sensitive, even for low amounts of RNA, delivers high yields and is superior to leading competitor enzymes in the production of fulllength cDNA, especially for long templates. NovaScript III is robust, easy to use and works well under a wide range of temperatures. To conclude, NovaScript III is an all-purpose reverse transcriptase with excellent features and is superior than other reverse transcriptases available commercially.
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NOVASCRIPT III RNase H minus Reverse Transcriptase
Storage Conditions: |
To be Stored at – 20ºC. NovaScript III will remain stable if stored as specified.
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Shipping Conditions:
On Dry Ice (-20ºC)
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Concentration:
200u/:l
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Components: |
| NOVASCRIPT III Storage Buffer |
5x Reaction Buffer |
| 25 mM Tris-Cl (pH 7.9) |
250 mM Tris-cl (Ph 8.3) |
| 100 mM NaCl |
350 mM KCl |
| 1mM EDTA |
15 mM MgCl2 |
| 5Mm DTT |
50 mM DTT |
| 0.1% Triton X-100 |
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| 50% glycerol |
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Unit Definition: |
One unit catalyses the incorporation of 1 nmole of dTTP into acid-insoluble material in 10 minutes at 37°C in 50 mM Tris-HCl, pH 8.6, 40mM KCl, 1 mM MnSO4, 1 mM DTT, and 0.5 mM [3H]TTP, using 200 μM oligo(dT)12-18-primed poly(A)n as template.
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Protocol for first-strand cDNA synthesis with NovaScript III RNase H minus Reverse Transcriptase |
A. Prepare a mix, containing:
1. Template RNA: |
| Total RNA |
0.5 – 5 :g |
| or mRNA |
0.01 – 0.5 :g |
| or Specific RNA |
Up to 0.5 :g |
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| NB: The amount of total RNA or mRNA required for the reaction depends on the level of expression of the gene encoded. |
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| 2. Primer: |
| Oligo (dT)18 |
0.5 :g |
| or Random hexamer |
0.2 :g |
| or Specific oligo |
5-20pmol |
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| 3. Deionized water (nuclease-free) up to 12μl total |
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| C. Add the following: |
| RNase inhibitor (optional) |
10 units |
dNTP Mix
• 40mM dNTP mix (10mM each)
• or 100mM dNTP mix (25mM each) |
1 :l
0.4 :l |
| 5x Reaction Buffer (provided) |
4 :l |
| Deionized water (nuclease-free) |
up to 19.75:l total when using NovaScript III at 200u/:l |
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| NB: We recommend a total dNTP concentration of 2 mM but the concentration can be decreased to 0.5 mM during the optimization of the reaction. |
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D. Mix by pipetting. Add 0.25-1.0 μl of NovaScriptIII at 200 u/μl.
E. Incubate at 37-42°C for 60 min |
NB: - If random hexamer oligos are used, an initial 10-minute incubation at 25°C is recommended.
- Incubation at 37-42°C is desirable for generating longer(>1000 bases) cDNAs, however, the use of higher incubation temperatures up to 50°C can increase the yield of cDNA synthesised (<1000 bases), especially in cases of complex RNA secondary structures
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F. Stop the reaction by heating at 70°C for 10 minutes,or by adding of an equal volume of 10 mM EDTA (pH 7.0). Chill on ice.
G. Use the cDNA synthesized in subsequent amplification reactions without any additional purification |
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