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- PCR
- Real-Time Quantitative PCR
- SYBR Green Detection
- DNAWe use Perfecta Sybr Green Fastmix. We are starting to use uMELT software to predict qPCR melt curves. Could you please provide me with this info so that I can accurately use uMELT? [Mono+] : Concentration of monovalent cations in solution (mM).The PerfeCta SYBR Green Fastmix has 50 mM salt and 2.2mM Free Mg at 1X. However, the co-solvents and the concentration of SYBR green I dye in the mastermix also affect amplicon Tm, so you may still not be able to accurately use uMELT. Our R&D has found uMELT mostly useful for predicting multiple melting domains in an amplicon.What is the amount of cDNA used per TaqMan or SYBR qPCR Assay?Suggested input quantities of template are: cDNA corresponding to 1 pg to 100 ng of total RNA; 100 pg to 100 ng genomic DNA. For more information, please consult the PerfeCta qPCR Supermixes or PerfeCta SYBR Green Supermixes protocols.
- DNA
- Probe-based DetectionWhat are the advantages of PerfeCTa qPCR FastMix vs PerfeCTa qPCR Supermix?Amplification can be run in about X minutes, at least 3X faster than standard runs with the PerfeCTa qPCR Supermix, with comparable results. For more information on using these reagents, please consult the PerfeCTa qPCR FastMix and PerfeCTa qPCR Supermix Protocol.What is the amount of RNA used per TaqMan® or SYBR qPCR Assay?Suggested input quantities of template are: 1 pg to 1 μg total RNA; 10 fg to 100 ng poly A(+) RNA; 10 to 1x108 copies viral RNA. For more information, please consult the qScrip One-Step qRT-PCR Kits protocols.
- RNAWhat is the amount of RNA used per TaqMan® or SYBR qPCR Assay?Suggested input quantities of template are: 1 pg to 1 μg total RNA; 10 fg to 100 ng poly A(+) RNA; 10 to 1x108 copies viral RNA. For more information, please consult the qScrip One-Step qRT-PCR Kits protocols.
- SYBR Green Detection
- Real-Time Quantitative PCR
- Next Generation Sequencing (NGS)
- DNAWhat is the recovery rate with sparQ PureMag Beads?The recovery rate depends on many factors, including the fragment size, sample volume, sample concentration, and elution volume. Up to 90% recovery rate can be expected under the optimal condition. The yield is also correlated with the ratio of beads to sample, and the lower ratio usually results in lower yield. When the ratio reaches to 1.8X, it generally produces maximal recovery.
- DNA
- Reverse Transcription
- First-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.Does the qScript™ cDNA Synthesis Kit include an RNase Inhibitor like the qScript™ cDNA SuperMix?Both kits contain an RNase inhibitor protein. qScript™ cDNA SuperMix is provided as a single tube reaction of 5X concentrated master mix. It provides all necessary components for first-strand synthesis including: buffer, dNTPs, MgCl2, primers, RNase inhibitor protein, qScript reverse transcriptase and stabilizers (except RNA template) . In the case of the qScript™ cDNA Synthesis Kit, Rnase inhibitor is premixed with the reverse transcriptase and is provided as a concentrated enzyme.What is the qScript™ Flex cDNA Kit? How is it different from the qScript™ cDNA Synthesis Kit?The qScript Flex cDNA Synthesis Kit is an easy-to-use and highly efficient kit for the synthesis of first-strand cDNA that enables your choice of cDNA priming method. Various RT-PCR applications may require different priming strategies for optimal performance and this kit provides optimized reagents for priming with oligo(dT)20, random primer, gene-specific primer (GSP), or any combination thereof. Therefore, qScript™ Flex cDNA Kit is ideal for developing the best protocol for your application. The qScript™ Flex cDNA Kit also allows the use of Oligo dT or gene-specific primers (GSP) for long RT-PCR of RNA targets up to 12 kb. The qScript cDNA Synthesis Kit consists of an optimized blend of random and oligo(dT) primers. It provides robust, consistent and unbiased first-strand synthesis over a broad range of RNA template concentrations. qScript™ cDNA Synthesis Kit is well suited for short RT-PCR apllications (i.e. qRT-PCR) and is not recommended for amplification of RNA's longer than 1kb. When using a mixture of the Oligo dT and Random Primer solutions, the qScript™ Flex cDNA Kit achieves first-strand synthesis over a broad range of RNA template concentrations and its performance is comparable to that of the qScript™ cDNA Synthesis Kit. With both kits, the resulting cDNA product is directly compatible with current real-time RT-PCR methods or end-point RT-PCR.
- First-Strand cDNA Synthesis
- Sample Preparation
- DNAWhat is the shelf life of the Extracta DBS product?The shelf life is 2 years stored at 2-8°C.
- DNA
- Real-Time qPCRWhat is the amount of RNA used per TaqMan® or SYBR qPCR Assay?Suggested input quantities of template are: 1 pg to 1 μg total RNA; 10 fg to 100 ng poly A(+) RNA; 10 to 1x108 copies viral RNA. For more information, please consult the qScrip One-Step qRT-PCR Kits protocols.
- Miscellaneous Questions
- What is the purpose of the 25C incubation step when using Q-Script-RT during cDNA synthesis?The purpose of the 25C incubation step during cDNA synthesis is to allow annealing and extension of the random primers. Since the primers are short, a lower temperature is required. Omission of this step will result in inefficient cDNA synthesis.
- How are PerfeCTa® microRNA assays designed?PerfeCTa microRNA assays are designed with primer design software according to the following criteria: • Optimized primer Tm designed to match the Universal PCR Primer • Universal cycling conditions to ensure robust amplification for all assays in profiling experiments • No self-complementarity or primer dimer artifacts with the