Kit TURBO DNA-free™

Yes, while a single DNase treatment is typically sufficient for the vast majority of DNA removal needs, a second treatment option is available. Please note that sequential TURBO DNA-free treatments require a special buffer for the second digestion step; the 10X TURBO DNase buffer provided in the kit cannot be used for this purpose. A unique digestion buffer for the second DNase reaction must be used to minimize the carryover of salt into the PCR step of RT-PCR. Otherwise, the limit of detection of targets in RT-PCR may be compromised.

Please see the second digest protocol below:

Before you begin:
Prepare the 10X TURBO DNA-free Second Digest Buffer. This buffer contains: 200 mM Tris-HCl pH 7.5, 100 mM MgCl2, and 5 mM CaCl2, prepared with nuclease-free H2O. The final pH of the solution should be 7.5. If the pH is not 7.5, add NaOH or HCl as necessary, but do not introduce more than 50 mM of total monovalent salt to the 10X buffer when adjusting the pH in this way.

Protocol:
After the DNase Inactivation Reagent treatment step in Section E.5 of the TURBO DNA-free Kit manual (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/1907M_turbodnafree_UG.pdf), remove a volume of the RNA sample without disturbing the pellet, and transfer it to a new 0.5 mL tube.

Add 0.1 volume of 10X TURBO DNA-free Second Digest Buffer, and 1 µL TURBO DNase to the RNA sample, mix gently, then continue with the standard protocol as described in Section E.2.

The RNA sample is now ready for downstream applications. However, we recommend consideration of the following points:

- For RT-PCR applications, we recommend that the volume of RNA that has been sequentially treated with TURBO DNA-free comprise no more than 20% of the final one-step RT-PCR reaction volume. If the amount of RNA passed into the RT-PCR reaction is too low, then consider increasing the RT-PCR volume to 50 µL or more, or performing a two-step reaction. For example, the RNA volume can be increased up to 40% of the final reverse transcription reaction volume in two-step applications.

- We have observed in-house that some RT-PCR amplicons are more sensitive to salt than others. Restricting the volume of the treated RNA sample to <20% can help to minimize these effects.

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 Invitrogen 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.

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

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

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

Most of our DNase I products are guaranteed free of RNase activity. However, please note that Cat. No. 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.

No. We do not offer the DNase Inactivation Reagent included in the TURBO DNA-free Kit (Cat. No. AM1907) as a standalone product. It is only available as part of the kit.

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