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phenol.
EDTA chelation of cations: The addition of EDTA to DNase digestion reactions chelates ions in the digestion buffer, that are required for DNase I activity. The DNase I can then be safely heat inactivated without loss of RNA. However, Mg2+ is needed for enzymatic activity of both the reverse transcriptase and the thermostable DNA polymerase. Thus the chelation capacity of the EDTA must be saturated with additional ions prior to subsequent enzymatic reactions. This can make the assembly of a simple reaction quite complicated.
RNA purification: Some filter-based RNA isolation methods treat with DNase directly on the filter after binding of the lysate. This treatment may not completely eliminate contaminating DNA because the DNase will not be in an optimal environment for digestion (traces of lysis solution and other contaminants may interfere with optimal digestion). Alternatively, RNA preparations that have been treated with DNase in solution, can be purified away from DNase over such columns. Although this technique adequately removes DNase from the prep, it requires both an extra step, and expensive materials.
RNA Isolation for RT-PCR
With the RNAqueous-4PCR Kit, you can isolate RNA free
of genomic DNA contamination from samples as small as 100 cells or 1 mg
of tissue. The kit contains reagents for the phenol-free isolation of
RNA, and reagents to remove contaminating DNA. The kit also contains plastic
pestles designed for disruption of small tissue samples, using microfuge
tubes as mortars. The RNAqueous procedure involves disrupting tissues
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