How should I purify my samples? How should I remove DNA or RNA contamination?

Bead based protocols (e.g. Ampure XP, RNAClean XP) and spin column based protocols (Qiagen, Zymo, NorgenBiotek, …) tend to be the most efficient ways to remove chemical contaminants.  For Illumina sequencing we suggest spin column based solutions as the most reliable option. Genomic DNA cleaned up with a spin column further has the advantage that it will always dissolve well.

Multiple protocols are available to remove DNA or RNA contaminations. Please find our suggestions for affordable solutions for Illumina sequencing below.

RNA samples need to be DNA-free  (the RNA isolation protocol should always include a DNAse digestion step; in problematic cases you could use RNA-clean & concentrator kits with DNAse).  On an agarose gel, DNA contamination will be visible as a smear of band of fragments considerably larger than the RNA (>10 kb).  On the Bioanalyzer RNA-chips DNA contamination will be visible in the size range from 4kb to 10 kb.
In case you are using a Trizol protocol for the RNA extractions, we would highly recommend to clean up the samples afterwards with a spin column kit (e.g. RNA-clean & concentrator kits) to make sure to remove any phenol traces.

DNA samples need to be RNA-free  (the DNA isolation protocol should always include a RNAse digestion step; in problematic cases we recommend to use RNAse I  (e.g. adding 1 ul RNAse I to your sample and incubate at 30 degrees C for 20 minutes).   RNAse I does not require a special buffer ( it works in TE buffer)  and can be completely inactivated by heating at 70°C for 15 minutes.  Thus, a removal of the enzyme and of a buffer can be avoided in many cases.  If you want to perform a cleanup, Ampure beads (or similar) or DNA-clean & concentrator kits will work fine.  DNA samples can be QC-ed easily by agarose gel electrophoresis and ethidiumbromide staining. The stain will make both DNA and RNA visible.  RNA will run as an halo-like smear in the range of 50- to 200 bp.


Category: 03 Sample Preparation

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