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SuperScript™ IV First-Strand Synthesis System, Each - FAQs

SuperScript™ IV First-Strand Synthesis System, Each - FAQs

View additional product information for SuperScript™ IV First-Strand Synthesis System - FAQs (18091150, 18091050, 18091200, 18091300)

12 product FAQs found

Is it normal to see a band in the SuperScript IV First-Strand Synthesis System (Cat. Nos. 18091050, 18091200) "No Reverse Transcriptase (RT) Control"?

Yes. The SuperScript IV First-Strand Synthesis System (Cat. No. 18091050, 18091200) "No Reverse Transcriptase (RT) Control" will often display a level of amplification because of residual genomic DNA in the RNA preps. In general, you do not need to worry if a faint band shows up on a gel, as long as the intensity of the positive control is much higher.

Find additional tips, troubleshooting help, and resources within our PCR and cDNA Synthesis Support Center.

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.

Find additional tips, troubleshooting help, and resources within our PCR and cDNA Synthesis Support Center.

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.

Find additional tips, troubleshooting help, and resources within our PCR and cDNA Synthesis Support Center.

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).

Find additional tips, troubleshooting help, and resources within our PCR and cDNA Synthesis Support Center.

For real-time PCR applications, why do you recommend the SuperScript IV VILO Master Mix over the SuperScript VILO Master Mix?

For real-time PCR applications we recommend the SuperScript IV VILO Master Mix for superior performance and resistance to inhibitors.

Can Superscript IV RT be used at higher reaction temperatures to reverse transcribe targets with secondary structure?

SuperScript IV RT sustains 100% activity up to 56.4 degrees C and 70% activity up to 65 degrees C. The enzyme's ability to function at higher temperatures enables the reverse transcription of RNA targets with strong secondary structure.

What is the longest cDNA product that Superscript IV RT can synthesize?

We have found that SuperScript IV RT was able to produce a 12.3 kb cDNA product while all other reverse transcriptases tested at the same time produced smears and smaller products, using a gene-specific reverse transcription primer.

For more information about the performance of SuperScript IV, please refer to the whitepaper Whitepaper: SuperScript IV Reverse Transcriptase.



Find additional tips, troubleshooting help, and resources within ourReverse Transcription and RACE Support Center.

Which inhibitors has Superscript IV RT been tested with for superior performance?

SuperScript IV RT functions exceedingly better than all tested enzymes in the presence of TRIzol Reagent, ethanol, SoluLyse reagent, guanidinium salts, LiCl, ammonium acetate, hematin, and humic acid. SuperScript IV RT is also able to perform in the presence of formalin, paraffin, and isopropanol.

For more information about the performance of SuperScript IV, please refer to the white paper Whitepaper: SuperScript IV Reverse Transcriptase.

Can I use the Invitrogen SuperScript III RT/ Invitrogen SuperScript II RT 5X first-strand buffer with Invitrogen Superscript IV RT?

We do not recommend using SuperScript III RT/ SuperScript II RT 5X first-strand buffer with Superscript IV RT. For optimal performance of Superscript IV RT, we recommend using it with the 5X RT buffer supplied in the kit.

What is the composition of the 5X RT buffer supplied with Invitrogen SuperScript IV RT?

The composition of the buffer is proprietary.

What are the unique features of Invitrogen Superscript IV Reverse Transcriptase? How does it compare to Invitrogen Superscript III Reverse Transcriptase?

While retaining all the performance benefits of SuperScript III RT, SuperScript IV RT has the following additional benefits:

- Significantly improved resistance to a variety of inhibitors that can interfere with cDNA synthesis
- Robust and specific cDNA synthesis across a wide range of sample types
- Increased reproducibility
- A faster reverse transcriptase reaction time that reduces the incubation time from >50 minutes to 10 minutes
- Increased thermostability
- Significantly better processivity