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DNA-free A NEW Method to Remove DNA

inactivation of this enzyme is problematic; DNase removal methods can be inconvenient, ineffective and even detrimental to RNA integrity.

Commonly used methods for removal or inactivation of DNase after digestion include: heat inactivation, proteinase K treatment followed by phenol:chloroform extraction, chelation of essential ions with EDTA, and purification using a glass-filter binding method such as RNAqueous (see the sidebar at right, "RNA Isolation for RT-PCR). Each of these inactivation or removal methods has its drawbacks.

Heat inactivation: Probably the most common method of DNase inactivation is heat treatment, typically for 5 minutes at 75C. Although this method appears straightforward, the divalent cations in the DNase digestion buffer can cause (chemically-induced) strand scission of RNA when heated. Studies at Ambion have shown that much of an RNA sample is destroyed when heated to 80C for 5 minutes in the presence of 2.5 mM MgCl2 and 0.1 mM CaCl2 (salts typically found in DNase I digestion buffer).

Proteinase K treatment and organic extraction: Proteinase K treatment followed by phenol:chloroform extraction is probably the most rigorous method for DNase inactivation and removal, but it is time-consuming, and organic extractions often cause some sample loss. Sample loss can be minimized by back extraction of the phenol:chloroform phase, but this adds another step to an already time-consuming procedure. Additionally, many people prefer to avoid working with hazardous
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