How do I remove DNase from an RNA sample?

Product FAQ

Answer

DNase can be removed by the following methods: acid phenol:choloform extraction, lithium chloride precipitation, EDTA, and heat inactivation, or using our MegaClear™ Transcription Clean-Up Kit (Cat. No. AM1908).

Alternatively, our Ambion® TURBO DNA-free™ Kit (Cat. No. AM1907) is supplied with a DNase inactivation reagent that can be used to remove the DNase, as well as divalent cations such as magnesium and calcium, which can catalyze RNA degradation when RNA is heated with the sample.

Answer Id: E7822

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5ea154ebedd63e2ce09be299d2ade03b_FAQ

Do I need to treat my cDNA sample with DNase when used with qPCR experiments? If so, how should I do it?

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Answer

DNase treatment would depend on how your assay or primers were designed. If the probe or primer sits on an exon-exon junction, then the design is such that it will not amplify gDNA. In these cases you do not have to DNase-treat your sample to remove contaminating gDNA. However if, for example, your assay or primers are designed within a single exon, then you will want to treat your RNA with DNase. Our TURBO DNA-free™ Kit (Cat. No. AM1907) is a good option for this.

Answer Id: E7480

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294ea90f734ee8e21d7f932646bd5db7_FAQ

Are your DNase I products RNase-free?

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Answer

Most of our DNase I products are guaranteed free of RNase activity. However, please note that product 18047-019 is not tested for RNAse and is recommended primarily for protein applications. The other products are suitable for removing DNA from both RNA and protein preparations, for nick translating DNA, and for generating random fragments of DNA. For more demanding RT-PCR applications, we recommend using DNAse I, Amplification Grade.

Answer Id: E2942

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808eac3cdf97d0f48a0bf43c48ce2dbd_FAQ

How can I inactivate DNase I?

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Answer

Add of 1 µl of 25 mM EDTA solution to the reaction mixture in 10 ul reaction with 1 unit DNase I, Amplification Grade (or 1:1 molar ratio of Mg++:EDTA) to chelate the Mg++ ions in the DNase I buffer. Heat for 10 min at 65°C.

Please Note: It is vital that the EDTA be added to at least 2 mM prior to heat-inactivation to avoid Mg dependant RNA hydrolysis.

DNA-free and Turbo-free versions of DNase I can be inactivated with included DNase Inactivation Reagent.

Answer Id: E2945

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631af69c0bfcae0600b424e77e6e2cc6_FAQ

Can I perform a second digest by treating with the TURBO DNA-free™ Kit?

Product FAQ

Answer

Yes, while a single DNase treatment is typically sufficient for a vast majority of DNA removal needs, a second treatment option is available. Please see the protocol here (http://www.lifetechnologies.com/us/en/home/references/ambion-tech-support/nuclease-enzymes/turbo-dna-free-second-digest-protocol.html).

Answer Id: E7823

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