DNA-free™ Kit From Ambion

DNase treatment and removal reagents are designed for the removal of contaminating DNA from RNA samples and for the removal of DNase after treatment. Since no method of RNA isolation can reliably produce RNA that is completely free of contaminating DNA, many researchers choose to treat their RNA samples with some kind of DNA degradation enzyme to remove trace levels of DNA.

The DNA-free™ Kit from Ambion comes in the size of 50 rxns, complete with RNase-free DNase I, an optimized 10X Reaction Buffer, and a novel DNase Removal Reagent, with or without a user manual, which can also be downloaded from an online source. It is also available as a Turbo DNA-free™ Kit, which utilizes an Ambion-engineered hyperactive DNase. Personally, I was satisfied with the performance and the removal of DNA contamination using the standard DNA-free™ Kit, therefore, I stuck to the kit containing the wildtype DNase.

I use this kit on a regular base for removing DNA from RNA samples after isolating the RNA from nucleated cell samples for downstream applications such as RT-PCR. The second-most application for which I use this kit is to remove DNA from RNA samples after in vitro transcription reactions.

One of the major advantages of this kit is the easy removal of the digestion enzyme. In contrast to other approaches which require phenol/chloroform extraction, alcohol precipitation, heat inactivation, or the addition of EDTA, the sample is incubated with an inactivation reagent which will be centrifuged out together with the DNase as well as divalent cations, such as magnesium and calcium.

The beauty of the kit is it’s the simple protocol as well as the short experimental time. Add the DNase buffer as well as the rDNase to the sample. The protocol mentions the possibility of adding up to 3 ul of enzyme to the mix. Having dealt with heavily contaminated samples, I used this higher amount of DNase and got complete removal of DNA from my RNA of interest. The sample gets incubated at 37ºC for 30 min. The next step is the addition of the DNase inactivation reagent which is some kind of resin, and therefore needs to be resuspended thoroughly before use. After a short incubation with intermitting mixing of the sample, just spin the sample down for 1.5 min. and transfer the RNA to a fresh tube. Regardless of all the advantages of this kit, this step is one of the drawbacks of the technique. The inactivation resin doesn’t form a very firm pellet which makes it hard to recover all the fluid with the cleaned RNA, therefore, always resulting in some loss of RNA. The company recommends avoiding the introduction of inactivation reagent into the cleaned RNA, because it might sequester divalent cations and change the required buffer conditions for downstream applications.

Overall, I think the kit is working very well, is easy to use and performs very fast.