DNase I, Amplifikationsstufe

RNAse-free DNase treatment of the RNA can reduce DNA to undetectable levels. We recommend using our DNase 1, Amplification Grade (Cat. No. 18068015).

We do not test for protease activity as part of our QC but there is PMSF (a protease inhibitor) in the storage buffer. Furthermore, in the preparation of DNase I, we use a soybean trypsin inhibitor column to remove proteases.

DNase I treatment is optional, and one has to consider individual experimental design.

Potential disadvantage of omitting the DNase I step: you may get amplification from genomic DNA. If you omit this step, you will need to include a no RT control and design primers that will not amplify genomic DNA, like those spanning two different exons or exon-exon junctions.

Potential benefit of omitting the DNAse I Step:
saves time; consumes less reagent, saves pipetting steps, and reduces RNA loss (important for precious samples).

Protocol for DNAse I treatment:
Combine 1 µg total RNA, 1 µL 10X DNAse I buffer (200 mM Tris-HCl (pH 8.4), 500 mM KCl, 20 mM MgCl2), 1 µL Amplification Grade DNAse I (1 unit/µL), and DEPC-treated water to 10 µL. Incubate for 15 min at room temperature. Inactivate by adding 1 µL of 25 mM EDTA and heat for 10 min at 65 degrees C.
Note: 1 U of DNAse I should be enough to treat up to ~10 µg of RNA.

To locate the manual for Amplification Grade DNAse I, search www.thermofisher.com with the Cat. No.18068-015. The manual will be one of the links on the product page.

The Amplification Grade DNase I (Cat. No. 18068-015) is subjected to an extra final HPLC purification step to remove traces of RNases. The Amplification Grade DNase I is supplied as 1 unit/µL and comes with 10X buffer (200 mM Tris-HCL pH 8.4, 20 mM MgCl2, 500 mM KCl) and a vial of 25mM EDTA.

In RT-PCR, a large excess of Amplification Grade DNase I could be used to digest an RNA template without degradation of the RNA (in-house data). Use Amplification Grade DNase I to remove genomic DNA carryover in RNA samples prior to RT-PCR.

The regular DNase I is supplied at 5-15 mg/mL (50-375 U/µL) and does not come with its own buffer.

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.