UM_TotalRNA_NucleoZOL(17)

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6 Appendix

6.1 Digestion of residual DNA solution

NucleoZOL efficiently removes DNA when processing samples according to the standard protocol, resulting in minimal residual DNA in the purified RNA. Residual DNA will not be detectable in most downstream applications. If large samples or samples with high levels of DNA are processed, it may be difficult to remove all traces of DNA. The amount of residual DNA depends on the sample type, amount, DNA content and the detection sensitivity of the method used to analyze residual DNA. A typical example is an RT-PCR reaction in which the primer molecules do not differentiate between cDNA (derived from RNA) and contaminating genomic DNA. The effect is prominent if

high copy number targets are analyzed (e.g., multi gene family, mitochondrial, plastidal or plasmid targets (from transfections)the target gene is of a very low expression levelthe amplicon is relatively small (< 200 bp).

DNA digestion in solution can efficiently destroy contaminating DNA. However, stringent RNase control and subsequent repurification of the RNA (in order to remove buffer, salts, DNase and digested DNA) are usually required. High quality, RNase-free, recombinant rDNase (REF 740963, see ordering information 6.3) facilitates such a digestion in solution in order to remove traces of contaminating DNA.

A) Digest DNA (Reaction setup)

Add 6 μL Reaction Buffer for rDNase and 0.6 μL rDNase to 60 μL eluted RNA. (Alternatively, premix 100 μL Reaction Buffer for rDNase and 10 μL rDNase and add 1/10 volume to one volume of RNA eluate). Gently swirl the tube in order to mix the solution. Spin down gently (approx. 1 s at 1,000 x g) to collect every droplet of the solution at the bottom of the tube.B) Incubate sample

Incubate for 10 min at 37 °C.

C) Repurify RNA

Repurify RNA with a suitable RNA cleanup procedure: NucleoSpin® RNA Clean-up, NucleoSpin® RNA Clean-up XS kits (see ordering information 6.3), or by ethanol precipitation.

Ethanol precipitation, exemplary:Add 0.1 volume of 3 M sodium acetate, pH 5.2 and 2.5 volumes of 96–100 % ethanol to one volume of sample. Mix thoroughly. Incubate several minutes to several hours at -20 °C or 4 °C.

Centrifuge for 10 min at maximum speed.Wash RNA pellet with 70 % ethanol.

Dry RNA pellet and resuspend RNA in RNase-free water.