Nucleases contamination

Pellet cells for 5 min in two 50 mL Falcon tubes (e.g., 2000 rpm in a Beckman J-6M swinging bucket centrifuge with Beckman JS-4.2 rotor). (We use sterile Falcon tubes (BD Biosciences, San Jose, CA, cat. 352070) to minimize the chance of nuclease contamination.) The supernatant is removed. Optionally, one pellet can be resuspended in equal volume of LB (1% Bacto Tryptone, 0.5% yeast extract and 0.5% NaCl in water) and 60% glycerol, divided into multiple fractions and stored at —80 °C for future use. The other pellet is resuspended in 25 mL at 37 °C LB, then add into 2 L LB containing 2 mL of 50 mg/mL Carbenicillin and 2 mL of 10 mg/mL Chloramphenicol. Grow at 37 °C until the ODgoo is close to 1,... 

Sterile microcentrifiige tubes and pipet tips should be used for all reactions. These items should be sterilized by gamma irradiation rather than steam autoclaving. Sterile tips and tubes are available commercially (e.g., Bio-Rad [tips] and Sarstedt, Germany [tubes]). Sterile, cot-ton-plugged pipet tips are recommended to prevent carryover between samples and nuclease contamination from the pipetor. Pipet tips should not be used more than once. 

Unlabeled RNA is added as a competitive inhibitor, or as a nonspecific carrier that may interfere with nuclease contamination or nonspecific RNA-protein interactions. Specific inhibitors for RNA-binding reactions will typically be derived from in vitro transcription using any of a number of commercially available in vitro transcription kits. These will typically be added in 1- to 100-fold molar excess over the radiolabeled... 

Product manufacture entails viral vector propagation in a suitable animal packing cell line (known as HEK 293). After cell recovery and lysis, the crude product is clarified by filtration and concentrated by ultrafiltration. The product is then treated with a nuclease preparation in order to degrade contaminant DNA and further downstream processing entails multi-step high-resolution column chromatography (see also Figure 14.7). 

Nuclease digestion of targets by endogenous RNase or DNase or by contaminating RNase. 

More details concerning the properties of these contaminating enzymes may be found in reviews (17-21) and books (22-24) devoted to nucleases. 

Endotoxins are found in some bacterial sources, such as E. coli. For other products they are considered a contaminant that should not be present and can be controlled by adherence to good manufacturing practices (GMPs). Nucleic acids, once considered a significant risk, are now thought of as cellular impurities, and their removal should be validated [2,3]. Proteins that pose a potential risk (e.g., immunogenicity) include host cell proteins, aberrant protein product, proteins used in cell culture, and those associated with the process (e.g., protein A affinity ligands or nucleases employed to reduce viscosity). 

In the isolated perfused liver experiments, buffers containing no erythrocytes or serum proteins are used to examine the direct interaction of a gene drag with tissues and to avoid the interaction of a gene drag with blood components and possible contamination of nucleases. 

Another clear benefit of PNA probe chemistry is its exceptional stability. PNA molecules are highly resistant to both nuclease and protease enzymes, and are stable over a wider pH range than DNA or RNA molecules. Probe stability is especially important in diagnostic settings with potentially high amounts of contaminating enzymes, such as assays of minimally processed biological specimens or... 

Terminal heterogeneity at the labelled end due to action of contaminating nuclease in the restriction enzyme or in the labelling procedure... 

Care should be taken when preparing solutions that impurities are not introduced during weighing, pipetting or addition of solvent. That means that dean weighing vessels and spatulas should be used, and that pipettes, tips, or the inside of storage vessels should not come into contact with hands even clean skin contains surface fats, salts and enzymes, particularly nucleases, which can cause serious contamination. Solutions should not be left uncovered, and in particular, stock solutions should be stored where the risk of decomposition or contamination is minimised this usually means in the cool and dark, conditions which are found in a refrigerator. 

The conditions for proteolytic digestion may need to be varied, and it is advisable in the first instance to perform a series of digestions, varying the concentration of proteinase K from 1-20 mg/mL. In principle, any enzyme with proteolytic activity can be used, but remember that enzyme preparations may be contaminated with nuclease activities which could give false positive results. For this reason, pepsin or proteinase K are good choices, because the former is used at low pH where nucleases would not be active, and the latter is supplied in a highly purified form and is effectively free of nuclease activity. 

Nucleases are enzymes that hydrolyze one or more phos-phodiester bonds in nucleic acid polymers. Nucleases may require a free hydroxyl end (exonucleases), with specificity for the 3 or 5 end, or may act only on internal bonds (endonucleases). For example, some probe techniques are based on 5 -exonuclease activity that cleaves nucleic acids between two fluorescent labels. Nucleases can be DNA- or RNA-specific and may act on only double- or single-stranded polymers. For example, DNAse I digests double-stranded DNA (dsDNA) and SI nuclease acts only on smgle-stranded DNA (ssDNA). DNase I can be used to specifically degrade DNA in nucleic acid mixtures when only RNA is of interest. RNAses are very stable enzymes that are common laboratory contaminants. 

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