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View additional product information for SuperScript™ IV VILO™ Master Mix - FAQs (11766050, 11756500, 11766500, 11756050)
14 product FAQs found
Yes, the TaqMan Cells-to-CT Express Kit includes Express ezDNase which can be added to the Express Lysis Solution for removal of gDNA during cell lysis. Express ezDNase is a double-strand specific, heat-labile DNase that will only digest gDNA, making it compatible with the downstream reverse transcription reaction. The enzyme is automatically inactivated during a heat kill step included in the reverse transcription program. To avoid the detection of gDNA, we recommend using a TaqMan Gene Expression Assay specifically designed to span an intron-exon boundary. Such assays are designated with an "_m1" suffix. More information can be found here.
Find additional tips, troubleshooting help, and resources within our Real-Time PCR and Digital PCR Applications Support Center.
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.
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.
The ezDNase enzyme is highly specific for double-stranded DNA and does not cause damage to RNA and single stranded DNA. The high specificity of its action makes the deactivation step largely irrelevant in the subsequent RT reactions. ezDNase enzyme can be inactivated at 55 degrees C incubation for 2 min if needed.
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.