ezDNase™ Enzyme - FAQs

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

Find additional tips, troubleshooting help, and resources within our Nucleic Acid Purification and Analysis Support Center.

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. 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.

The composition of the ezDNA Enzyme buffer is proprietary.

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.

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.

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.