g DNA Contamination
• Filter-based purification methods yield RNA that is typically 1-10% DNA (based on real-time data).
• There is no RNA isolation method that generates RNA completely free of DNA contamination. Therefore you must DNase I treat your RNA samples
• A follow-up problem is how to get rid of the DNase I and divalent cations so they won’t be present during subsequent cDNA synthesis
DNase I treatment of RNA
increasing DNase I and time
Divalent Cations and Heat Degrade RNA
• DNase I Buffer with 0.1 mM CaCl2, 2.5 mM MgCl2
• Heated 90C, 5 minutes
• Run on formaldehyde agarose gel
• Don’t heat kill DNase I
• Unless you remove the divalents, don’t heat your RNA in the RT reaction
DNA-free™ DNase Treatment and Removal Reagents
• Optimized DNA digestion reagents
• Inactivates DNase without heating, phenol extraction or precipitation
• DNase Removal Reagent is added directly after DNase digestion:
inactivates DNase and removes divalent cations (Ca++, Mg++)
