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Reverse TranscriptionFirst-Strand cDNA SynthesisIs MMLV RT as stable in the qScript cDNA Supermix as in the qScript cDNA synthesis kit? Do you recommend storing these products at -80C?In the qScript cDNA synthesis kit, the RT/RNase inhibitor mix is provided as a concentrated enzyme separately from the 5X reaction mix. This enzyme is actually stable at -20C for a year. The cDNA SuperMix has a shelf life of 12 months at -20C. Neither product shows any diminished performance after storage in a REVCO at -70C to -80C; two years for the qScript cDNA Supermix and five years for the qScript cDNA synthesis kit. If you are looking to prolong shelf life, storage at -80C is best for the qScript cDNA Supermix. Storage at -20C is sufficient for the qScript cDNA Synthesis Kit.I am trying to amplify a very rare transcript. Can I scale up a cDNA synthesis reaction and use 2ug of mRNA?qScript cDNA SuperMix can easily handle up to 2 ug of total RNA in a typical 20-uL first-strand reaction without compromising cDNA synthesis efficiency. For the qScript cDNA Synthesis Kit we recommend doubling the reaction volume to 40-uL. Simply scale each component proportionally.Do the cDNA Supermix and the cDNA synthesis Kits have the same dynamic range in RNA quantity?Both qScript cDNA Supermix and qScript cDNA Synthesis Kits have a broad linear dynamic range. We recommend using them for first-strand cDNA synthesis using HeLa cell total RNA dilutions ranging from 1 µg to 1 pg. One tenth of each first-strand reaction should be used for qPCR amplification. Within this range of HeLa total RNA, qPCR results should be equivalent with both kits. qScript cDNA synthesis kit may have advantages over the qScript cDNA Supermix in certain situations such as working with small quantities of RNA. qScript cDNA Supermix outperforms the qScript cDNA Synthesis kit when starting with quantities of total RNA in excess of 100 ng. qScript cDNA SuperMix can easily handle up to 2 ug of total RNA in a typical 20-uL first-strand reaction without compromising cDNA synthesis efficiency. With the qScript cDNA Synthesis Kit we recommend doubling the reaction volume to 40-uL and simply scaling each component proportionally.What is the difference between qScript cDNA SuperMix and the qScript cDNA Synthesis kit?qScript cDNA SuperMix is the first ""true"" SuperMix format commercially available for cDNA synthesis. A single tube reaction, of 5X concentrated master mix, provides all necessary components for first-strand synthesis (except RNA template) including: buffer, dNTPs, MgCl2, primers, RNase inhibitor protein, qScript reverse transcriptase and stabilizers. In the qScript cDNA Synthesis kit the qScript RT (mixture of RNase inhibitor protein and qScript reverse transcriptase) is provided as a concentrated enzyme. A separate tube contains a solution of 5X concentrated reaction buffer, dNTPs, MgCl2, and primers. Aside from the inclusion of the enzyme and the stabilizers in the qScript cDNA SuperMix, its formulation is also slightly different from the qScript cDNA Synthesis formulation.What is the RNA input for qScript cDNA Supermix?The qScript cDNA Supermix provides for the quantitative conversion of 1µg to 10 pg total RNA to cDNA, with a reaction volume of 20 ul.In conventional cDNA-synthesis, an RNA-denaturation step is often included (typically 65 degrees C for 15 min.). Such a step is not mentioned in the protocol for the qSript cDNA SuperMix, is it not needed?We have not seen any need or benefit to including an RNA denaturation step with the qScript cDNA SuperMix. We have examined hundreds of different amplicons using real-time RT-PCR and have not seen any difference. However, there are exceptions to every rule – especially when it comes to RT-PCR. Disruption of RNA secondary structures, immediately prior to carrying out cDNA synthesis, is critical when using oligo-dT or gene-specific priming. Inclusion of the denaturation step is also important when using oligo-dT primer for generating long first strand products (i.e. amplification of full length genes). Our qScript Flex cDNA Synthesis kit allows the flexibility to choose the method of your choice (oligo-dT, random primer, gene-specific primer, or any combination thereof) for priming first-strand synthesis. We have been able to generate cDNAs that were over 15-kb long with this kit. How the denaturation step is carried out is very important. Often, incubation of RNA and primer in water alone at elevated temperatures can result in non-specific hydrolysis and degradation of the RNA. The qScript Flex kit includes a special additive in the primer solutions, as well as a separate tube of this enhancer solution for use with gene-specific primers, that protects the RNA from hydrolysis and facilitates efficient annealing of the primer(s). In the case of the cDNA SuperMix, it is possible that annealing of random primers to the RNA may facilitate disruption of secondary structure, and thus obviate the need for the denaturation step. Links to product information: qScript™ cDNA Synthesis, qScript™ cDNA, qScript™ Flex cDNA Links to product information: qScript™ cDNA Synthesis Kit:http://www.quantabio.com/pdf/manuals/95047%20(qScriptT%20cDNA%20Synthesis%20Kit%20PPS).pdf qScript™ cDNA SuperMix:http://www.quantabio.com/pdf/manuals/95048%20(qScript%20cDNA%20SuperMix%20PPS).pdf qScript™ Flex cDNA Kit:http://www.quantabio.com/pdf/manuals/95049%20(qScriptT%20Flex%20cDNA%20Synthesis%20Kit%20PPS).pdf