Ribonuclease DNase free

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... 

Na salts of ribonucleotide triphosphates (Roche or Sigma) bovine serum albumin RNase-free, 20 mg/ml (Roche) RNasin ribonuclease inhibitor, 40 U/ml (Promega) both bacteriophage T7 RNA polymerase and RNA Cap structure analog m7G(5/)ppp(5/)G are from BioLabs DNase-RNase-free (Roche) complete EDTA-free proteinase inhibitors cocktail (Roche) pyruvate kinase (PK) (Roche). 

The enzyme obtained by this purification procedure 11), when tested for contaminants under very stringent conditions, was found to be completely free from phosphatase, DNase, ribonuclease, and adenosine deaminase activities. 

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... 

Inhibitors of protein synthesis as chloramphenicol (Bernlohr and Novelli, 1963 Cornell and Snoke, 1964), puromycin, and tetracycline (Cornell and Snore, 1964) do not suppress the formation of bacitracin by either whole cells or protoplasts that had been cultured in or derived from Mn+ -sufficient environments. The quantity of antibiotic produced by subcellular extracts is reduced by the presence of ribonuclease (RNase) but not as profoundly as can be generally observed in a system of protein biosynthesis deoxyribonuclease (DNase) has no effect on bacitracin yield (Shimura et al., 1964). In contrast, the ability of cell free extracts to produce the antibiotic is completely destroyed by detergents as, for example, sodium deoxycholate and sodium la urylsulf ate apparently, intact lipoprotein membranes are essential for the biosynthetic process (Shimura etaL, 1964). 

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