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Invitrogen™ ezDNase™ Enzyme

Catalog No. 11766051
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Catalog No. 11766051 Supplier Invitrogen™ Supplier No. 11766051
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Description

SuperScript™ IV VILO™ Master Mix is a reaction master mix designed for fast, sensitive, and reproducible cDNA synthesis in RT-qPCR applications.

SuperScript™ IV VILO™ (SSIV VILO) Master Mix is a reaction master mix designed for fast, sensitive, and reproducible cDNA synthesis in RT-qPCR applications. Inclusion of ezDNase, further accelerates the RT-qPCR workflow through an extremely simplified genomic DNA removal step. This master mix is also available without ezDNase, which can be purchased separately.

Features of SuperScript IV VILO Master Mix include:
  • Super-fast RT reaction in just 10 minutes and gDNA removal in 2 minutes
  • Super-high yields—lower Ct values by more than 2 cycles ahead of all other reverse transcription reagents
  • Super-convenient one-tube cDNA reaction master mix for two-step RT-qPCR
  • Super-efficient reaction even with low template amounts and suboptimal purity samples
SSIV VILO master mix elevates the trusted VILO technology to the next level with the highly processive and thermostable SuperScript™ IV Reverse Transcriptase and further optimized buffer. These components enable efficient cDNA synthesis at higher temperatures and in less time. SSIV VILO master mix provides superior cDNA yield and sensitivity even with suboptimal purity or scarce templates. It is your new tool for more efficient and reproducible RT-qPCR.

For even better performance, some SuperScript IV VILO Master Mix products include ezDNase (also available as a separate purchase) for genomic DNA removal to further accelerate the RT-qPCR workflow. The extremely simplified genomic DNA removal step dramatically reduces the time of the entire reverse transcription protocol and reduces possible variation in gene expression due to RNA loss or damage during conventional DNase treatment.

SSIV VILO master mix gives you more cDNA in less time with less pipeting, less variation, less reaction inhibition, less gDNA interference, and with less sample than all the other cDNA synthesis kits or master mixes for RT-qPCR.

Source
Purified from E. coli expressing the pol gene of M-MLV, modified to increase thermostability and half-life, processivity, inhibitor resistance, and to reduce RNase H activity.

Performance and quality testing
Assayed for endodeoxyribonuclease, exodeoxyribonuclease, and ribonuclease, as well as yield and length of cDNA product.

For Research Use Only. Not for use in diagnostic procedures.

Specifications

Concentration 10X
For Use With (Application) RT-qPCR
Content And Storage • ezDNase Enzyme, 50 μL
• ezDNase Buffer (10X), 100 μL
• Nuclease-free water, 1.25 mL

Store at -5 to -30°C.
Shipping Condition On dry ice
Enzyme DNase
Compatible Buffer Reverse Transcription Reagents
Quantity 50 reactions
Product Type Enzyme
Product Line ezDNase
Sample Type RNA
Form Liquid
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Frequently Asked Questions (FAQs)

How can I quantify the ssDNA in samples that contain both ssDNA and dsDNA, using the Qubit ssDNA Assay Kit (Cat. No. Q10212)?

Unfortunately, it is not possible to efficiently measure the ssDNA content in a sample that also contains dsDNA with the Qubit ssDNA Assay Kit (Cat. No. Q10212). The dye in the Qubit ssDNA Assay kit binds to both ssDNA and dsDNA.

It is possible to treat the sample with a DNase that only degrades dsDNA, such as theezDNase Enzyme (Cat. No. 11766051), and measure the ssDNA concentration afterward. However, if all dsDNA is not successfully digested, there is a risk of overestimating the ssDNA content.

The following article describes a PCR-based method to detect ssDNA in dsDNA samples:

Quantitative amplification of single-stranded DNA (QAOS) demonstrates that cdc13-1 mutants generate ssDNA in a telomere to centromere direction
Does the TaqMan Cells-to-CT Express Kit contain reagents for the removal of genomic DNA (gDNA)?

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.

In your SuperScript IV RT protocols, there is no ezDNase inactivation step. Will ezDNase be active to affect RNA or the RT reaction?

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.

Which SuperScript IV RT format do you recommend for real-time PCR applications?

For RT-qPCR applications we recommend using the Invitrogen SuperScript IV VILO Master Mix. 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.
Are there any significant changes in the SuperScript IV RT protocol compared to the SuperScript III RT protocol?

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.
Can I get comparable cDNA yield and length using the SuperScript IV RT 10-minute protocol as when using the 50-minute protocol for SuperScript III RT?

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).
What is the composition of the ezDNase Enzyme (Cat. No. 11766051) buffer?

The composition of the ezDNA Enzyme buffer is proprietary.
Does the substrate of ezDNase Enzyme (Cat. No. 11766051) include DNA, in the context of a DNA-RNA hybrid?

The ezDNase Enzyme is a recombinant double-strand-specific DNase for the fast removal of contaminating genomic DNA from RNA preparations, and it is highly specific without impact on DNA in the context of a DNA-RNA hybrid.
Do you offer a one-step RT-qPCR kit containing SuperScript IV RT?

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.
Do I need to DNase treat my RNA when using SuperScript IV RT?

Yes, DNase treatment is highly recommended when any amplifcation will be performed after cDNA synthesis. Our ezDNase enzyme protocol is an extremely simplified genomic DNA removal step that occurs immediately before reverse transcription, and does not require re-purification of the RNA. This dramatically reduces the time of the entire reverse transcription protocol and reduces RNA loss or damage that can occur during conventional DNase treatment.

For Research Use Only. Not for use in diagnostic procedures.