Cells or tissues are first lysed and homogenized in Buffer RLT, which immediately inactivates DNases and RNases as well as proteases to ensure isolation of intact DNA, RNA, and proteins. The lysate is then passed through an AllPrep DNA spin column. This column, in combination with the high-salt buffer, allows selective and efficient binding of genomic DNA. The column is washed and pure, ready-to-use DNA is then eluted.
Ethanol is added to the flow-through from the AllPrep DNA spin column to provide appropriate binding conditions for RNA, and the sample is then applied to an RNeasy spin column, where total RNA binds to the membrane and contaminants are efficiently washed away. High-quality RNA is then eluted in RNase-free water.
Buffer APP, a novel aqueous protein precipitation solution, is added to the flow-through of the RNeasy spin column, and the precipitated proteins are pelleted by centrifugation. Intact total proteins are redissolved in an appropriate buffer and then ready to use in downstream applications. The kit includes Buffer ALO, which is compatible with SDS-PAGE, for dissolving the protein pellet. (See flowchart " AllPrep DNA/RNA/Protein procedure".)