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View additional product information for SuperScript™ IV VILO™ Master Mix - FAQs (11766050, 11756500, 11766500, 11756050)
27 product FAQs found
For RT-qPCR with FFPE samples, we recommend SuperScript IV VILO Master Mix. This mix offers enhanced performance with degraded RNA and inhibitor containing samples.
The Invitrogen ezDNase Enzyme is a novel DNase that is highly specific for double-stranded DNA. It has no activity on single-stranded DNA in RT reactions (primers or probes), or on RNA. The enzyme is also thermolabile—it is inactivated quickly at temperatures typical for the SuperScript IV RT reaction (e.g., 50 degrees C). The additional inactivation step is therefore not required for most RT-qPCR applications. If the RNA sample is to be used for RT-PCR of fragments ≥3 kb, incubate the sample for 5 minutes at 55 degrees C in the presence of 10 mM DTT to inactivate the enzyme.
Find additional tips, troubleshooting help, and resources within our PCR and cDNA Synthesis Support Center.
For RT-qPCR applications we recommend using the Invitrogen SuperScript IV VILO Master Mix (Cat. No. 11756050). The cDNA synthesis reaction setup with this master mix requires fewer pipetting steps and therefore reduces variation in the data. SuperScript IV RT, as a component of the master mix, offers the highest efficiency of cDNA synthesis step compared to competitors’ products.
Find additional tips, troubleshooting help, and resources within our PCR and cDNA Synthesis Support Center.
The only difference is that the incubation time for the reverse transcription reaction has been reduced to 10 minutes and inactivation time has been reduced to 5 minutes at 85 degrees C.
When compared with SuperScript III RT (and other manufacturers’ RTs) in a synthesis reaction for a 9 kb cDNA, SuperScript IV RT performed successful synthesis in just 10 minutes and did so with comparable (or improved) yield (as shown by gel band density).
Find additional tips, troubleshooting help, and resources within our PCR and cDNA Synthesis Support Center.
SuperScript IV RT is not recommended for one-step RT-qPCR applications. However, it is included in end-point one-step RT-PCR kits: https://thermofisher.com/ssiv-onestep
For one-step RT-qPCR, we recommend SuperScript III Flash Reverse Transcriptase or TaqPath RT-qPCR Master Mix products.
We recommend using T Cell Leukemia (Jurkat) Total RNA (Cat. No. AM7858) as a control. T cell Leukemia (Jurkat) Total RNA is derived from a cell line consisting of a single T cell clonotype. Running the Oncomine TCR Beta-LR Assay on Jurkat Total RNA should enable detection of a single clonotype.
Unlike other AmpliSeq panels, there are no BED files and no reference is needed. All downstream analysis takes place in Ion Reporter Software.
We have tested the Ion Library Equalizer Kit with this kit for library quantitation and it does function, but manually balancing library input into the Ion Chef instrument is preferred.
Torrent Suite Software 5.6 and Ion Reporter Software 5.6 are needed to generate reports. In Torrent Suite Software 5.6, two Planned Run templates are provided for use with the Oncomine TCR Beta-LR Assay.
The Oncomine TCR Beta-LR Assay has been developed and verified for use on the Ion GeneStudio S5 Series System. The assay can be used with the Ion PGM System, however, this instrument configuration is not supported.
Here are the components of the Oncomine TCR Beta-LR Assay:
- Oncomine TCR Beta‑LR Panel
- Ion AmpliSeq Library Kit Plus
- Ion Select Barcode Adapters
The primer pool is at 5X concentration.
The Oncomine TCR Beta-LR Assay enables long-read RNA sequencing (up to 400 bp amplicon length) for complete characterization of the T cell receptor Beta (TCR Beta) chain, including all three complementarity-determining regions (CDR1, 2, and 3) of the variable gene. This enables allele-specific interrogation of TCR Beta to potentially predict drug-mediated adverse events.
The primers are provided as a single pool.
Up to 16 samples can be multiplexed on an Ion 530 chip, up to 4 samples can be multiplexed on an Ion 520 chip, and up to 4 samples can be multiplexed on an Ion 318 chip.
A variety of research sample types including fresh-frozen tissue, whole blood, and sorted T cells are compatible with the Oncomine TCR Beta-LR Assay.
T cell repertoire sequencing helps:
- To characterize diversity and study features of T lymphocytes in blood and infiltrated tumors
- To study variable gene polymorphisms implicated in autoimmune disease or immune-mediated adverse events
- To study the production and function of therapeutic or engineered T cells
This kit is good for 24 samples.
The Oncomine TCR Beta-LR Assay is an RNA-based next-generation sequencing (NGS) research assay that enables the characterization of the T cell receptor Beta (TCR Beta) sequences including all three complementarity-determining regions (CDR1, 2, and 3) of the variable gene. The assay measures T cell repertoire diversity as well as clonal expansion, and allows for identification of allele-specific polymorphisms.
The SuperScript IV VILO Master Mix contains SuperScript IV RT, a ribonuclease inhibitor, and a helper protein. The helper protein helps to increase the efficiency of the reverse transcription reaction and thus improve cDNA yield. This master mix formulation allows for a simpler reaction setup with less pipetting and less variation between samples.
50 reaction packs (Cat. Nos. 11756050 and 11766050) include 200 μL of 'No RT' Control sufficient for 50 reactions. 500 reaction packs (Cat. Nos. 11756500 and 11766500) include 1 mL of 'No RT' Control sufficient for 250 reactions.
This is to help ensure that the bias from using either oligo (dT)18 or random hexamer primers alone will be eliminated. Although the data indicates that using random hexamers alone enables quantitation just as good as the combination of the two primers, this may improve data quality.
SuperScript IV VILO Master Mix often permits better cDNA synthesis results compared to other cDNA synthesis kits and master mixes. It has increased thermostability, allowing the RT incubation temperature to be raised up to 65 degrees C to deal with the GC-rich or structurally complex RNA templates.
The recommended reaction temperature for SuperScript IV VILO Master Mix is 50 degrees C. For GC-rich or structurally complex RNA templates, RT incubation temperature may be increased up to 65 degrees C.
SuperScript IV VILO Master Mix uses SuperScript IV Reverse Transcriptase (RT) in the optimized master mix formulation, whereas SuperScript VILO Master Mix and SuperScript VILO kits use SuperScript III Reverse Transcriptase. SuperScript IV RT has a significantly improved performance profile over SuperScript III RT in thermostability, sensitivity, yield, processivity, and the ability to synthesize cDNA efficiently from a wide variety of RNA samples, even those of suboptimal purity and integrity.
SuperScript IV VILO Master Mix is an upgrade from SuperScript VILO Master Mix and cDNA Synthesis Kit. It is designed to be used for cDNA synthesis in the two-step RT-qPCR applications for gene expression analysis.