RNase contamination, removal

RNase A Remove contaminated nucleic Johnson and Perry (1976)... 

Treatment with RNase A removes contaminating RNA and this can either be integrated into the purification procedure or performed after the DNA has been purified. Prior to use, ensure that the RNase A solution has been heat-treated to destroy any contaminating DNase. Alternatively, use DNase-free RNase purchased from a reliable supplier. 

Tissue sections are obtained from rats that are killed by decapitation. The brains (or other required tissue) are rapidly removed and frozen in isopentane that has been precooled (-30°C) on dry ice. Sections (14 pM) are cryostat-cut and mounted (two sections per slide) under conditions that minimize RNase contamination. Mounting sections close to the bottom of the subbed slides will conserve reagents required for double-label ISH. Multiple adjacent series of sections are obtained, depending on need. For example, it is advisable to take a series for each [ Sj-labeled riboprobe both alone and in combination with each appropriate digoxigenin-labeled riboprobe. Slide-mounted sections are sorted at -20°C (in cryostat or cold room) prior to the assay to obtain the required number of near adjacent series. Slides brought to room temperature cannot be... 

Verified DNA contamination in the RNA sample is removed using the RQl RNAse-free DNAse kit according to the manufacturer s instructions The RNA sample is diluted with RNAse-free water to a final volume of 8pL, which is mixed with 1 pL lOX reaction buffer and DNAse of 1 U/pg RNA. The mixture is incubated for 30 min at 37°C and the reaction stopped by adding IpL stop solution and incubating the mixture for 5min at 65°C. PCR is used to check for the complete removal of genomic DNA as described in step 10. Repeat steps 10 and 11 until the RNA sample is DNA free. 

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... 

RNase A (previously heat treated to remove contaminating deoxyribonuclease activity)... 

The isolation of DNA, which is suitable for digestion with restriction enzymes (which will be discussed in Section 6.1.7), is an essential requirement for both genetic engineering techniques as well as for the analysis of genetic makeup of industrial products. The isolation of DNA from various organisms requires specific protocols, and there are various companies that provide extraction kits. One vital element to remember (if one wishes to study with pure DNA preparations) is the contamination of the sample with other nucleic acids, namely RNA, as well as other macromolecules such as protein and polysaccharides. In order to avoid this, most extraction protocols include RNase for the removal of RNA, and proteinase K enzyme to remove protein contaminants [2]. 

DEAE-cellulose and/or SP-Sephadex chromatographies have proven useful in removing contaminating RNase T1 pi 2.9) and RNase T2 (pZ 5.0). Nuclease SI is stable when stored in 50% glycerol at -20°C. 

Enzymes should be handled with care to avoid contamination. Use a fresh pipette (or tip) for each aliquot that is removed from the parental vial. Never return unused material to the parental vial. Wear gloves to prevent contaminating the enzyme with proteases, DNases, RNases, and inhibitors often found on fingertips. Never pipette by mouth. 

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