DNase-free RNase, preparation

To isolate genomic DNA from E. coli, the cells are treated with lysozyme and then lysed by SDS in the presence of proteinase K. Proteinase K, which is active even in SDS solution, degrades proteins including nudeases. Cell debris, polysaccharides and unhydrolysed protein are removed by precipitation at room temperature with cetyltrimethylammonium bromide (CTAB). DNA is isolated from the supernatant by precipitation with alcohol. RNA can be removed from DNA preparations by incubation with DNase-free RNase. Further purification can be effected by a phenol/ chloroform/isoamyl alcohol (25 24 1) extraction, and/or by CsCl gradient centrifugation (see Sect. 4.3.4.2 ) to remove the remaining protein and RNA. 

A Working Solution of the stain should be prepared just before use, by combining 10 ml of 0.1% Triton X-100 in phosphate-buffered saline, 2 mg RNase (DNase-free, Sigma), and 200 pi PI solution (Sigma, 1 mg/ml in distilled water). Stock solutions of Triton X-100 and PI should be stored at 4°C. Centrifuge the ethanol-fixed cells (200 x g for 5 min) and discard the supernatant, removing it completely. 

Select slides with optimized proteinase K digestion and follow the steps below for DNase digestion. Prepare in an Eppendorf tube 1.0 pL RNasin (RNase inhibitor), 11.5 pL UV-irradiated dH20, 37.5 U DNase (RQl RNase free DNase, Promega Inc., Madison, Wl)/(1 U/pL) Total (per slide) 50.0 pL. 

Phenolic extraction of cell lysates is one of the oldest techniques in DNA preparation. Examples of these have been presented in Chapters 6 and 7. Single cells in suspension are lysed with a detergent, and a proteinase enzyme is used to break down the protein molecules. Non-nucleic acid components are then extracted into an organic (phenol-chloroform) solvent, leaving nucleic acids in the aqueous layer. Two volumes of isopropanol are added to the isolated aqueous phase to precipitate the high-molecular-weight nucleic acids as a white mass. These are then treated with DNase-free ribonuclease (RNase) to remove the RNA. This is followed by a second treatment with proteinase, phenol extraction, and isopropanol precipitation. After precipitation, the DNA is separated from the isopropanol by... 

Solutions (including doubled distilled water, buffer and culture medium) should be sterile and DNase- and RNase-free grade and stored at 2-8°C (+4°C) after opened. Unless stated otherwise, all solutions should be prepared in water that has a resistivity of 18.2 MU cm and total organic content of less than five parts per billion. This standard is referred to as doubled distilled water in this text. 

All solutions should be prepared by autoclaved DNase- and RNase-free vater. 

RNase A and DNase I (RNase-free, i.e., from Stratagene) for control preparations. 

Another issue that should be considered carefully is the purity of nucleic acids used at each step. Since it is known that RNA can self-cleave and ligate, DNA produced from PCR that is entering the next cycle of selection should be size-purified by native PAGE (Section 8.3.1.5). Finally, all solutions should be prepared from DNase/RNase-free reagents in DEPC H20, 0.2 pm filtered, and stored at 4 °C (buffers) or —20 °C (especially nucleotide solutions). 

Residual RNA in a DNA preparation can be removed by treatment with ribonuclease (RNase). RNase A, which is free of DNase, is available commercially, or the contaminant DNase in the crude RNase A solution can be heat inactivated by heating RNase A solution (10 mg/mL in 10 mM Tris-Cl, pH 7.5, 15 mMNaCl) at 100°C for 15 minutes [4], DNA solution in TE at a concentration of at least 100 pg/mL is treated with RNase to a final concentration of 1 pg/mL followed by incubation at 37°C for 1 hour [3], RNase... 

To reduce false-positive clones, mRNA should be prepared free from rRNA and genomic DNA as possible. We purify and concentrate mRNA by QuickPrep Micro mRNA purification Kit (Pharmacia) using glycogen as a carrier. Then, contaminated gemonic DNA is digested with RNase-free DNase I (Gibco-BRL). 

RNase A is known to be strongly adsorbed on Macaloid, a trade name for a negatively charged, purified clay mineral, heaorite (Na, Mg, Li-fluorosilicate). Macaloid has been used to block RNases during the preparation of RNA (31) and to eliminate contaminating RNases in preparing RNase-free DNases. 

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