DNase solution preparation

DNase I stock solutions are stored at -2O C (1 mg/ml) in 5- xl aliquots (each aliquot is used once). Variation in the activity of DNase I preparations is often observed and the exact amount needed to introduce the desired number of nicks should be determined for each enzyme batch. Sometimes template switches occur which will result in snap-back structures (zero-binding nucleic acid), which remain S, nuclease-resistant upon denaturation. Rigby et al. (1977) suggested that this effect was due to a differential loss of 5 - 3 exonuclease activity upon storage leading to a displacement of the nicked strand and a template switch from the complementary... 

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. 

Prepare a positive control bytreatingthe tissue or cells with DNase I for 10 min at 25°C. For negative control, incubate with staining solution only without the enzyme. 

Ribonuclease A (RNase A) is dissolved at 1 mg/ml in water, and stored at -20°C. To inactivate the possible contaminated DNase completely, RNase A solution is sometimes boiled once when prepared. 

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 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. 

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. 

Tissue Preparation 1. P2 Sprague-Dawley Rat pups. 2. Dulbecco Modified Eagle s Medium (DMEM), high glucose, no glutamine, no pyruvate (Cambrex). 3. Ca +-Mg free Hanks Balanced Salt Solution (Cambrex). 4. 2.5% trypsin solution (Cambrex) diluted 1 10 in HBSS for 0.25% final, aliquot and store at -20°C, stable for 1 year. 5. DNAse 1 (Boehringer Mannheim) prepare 1 mg/mL stock in PBS aliquot store at -20°C stable for 1 year. 6. Heat-inactivated FBS (Cambrex) aliquot and store at -20°C. 

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

The previous protocol can be interrupted at an intermediate stage after the first mitochondrial pellet (step 1 crude mitochondria pellet) or after the second one (step 3 pine mitochondria pellet). In both cases, mtDNA is extracted by SDS lysis of mitochiondria followed by phenol extraction (see below for a simplified preparation of phenol see also in section HI A the minipreparation of mtDNA with a similar principle). If mtDNA is contaminated, the optional step described above can be added or an alternative solution can be chosen DNase or digitonin treatment can be tried. These various protocols are described below. A protocol similar to the sucrose gradient but-involving Percoll density gradient, often of better efficiency, is described first in the next section. 

DNA catenanes were prepared by the addition of T4 DNA ligase to the mixture of linear DNA and nicked DNA as shown in Scheme 4.4. The linear and nicked DNAs were prepared by the addition of EcoRl or DNase I to a solution of pBR322 plasmid DNA, respectively. The advantage of using T4 DNA ligase is an irreversible recombination between the complementary ends of DNA, in contrast with the reversible one for topoisomerase I. Therefore we can expect an increase in the yield of DNA catenanes. Many slower migrated bands were observed in elec-... 

HeLa cells were permeabilized by the method of Thi Man and Shall [21]. Nuclei were isolated by the method of Hodge et al. [22]. Using 1% Nonidet P40 and 0.5% deoxy-cholate, nuclear matrices were prepared as described by Mariman et al. [8]. Briefly, the nuclei were subsequently digested with DNase I (500 /itg ml ) and with RNase A (100 jug ml" ) at 20°C for 15 min. After a sucrose step centrifugation, the nuclei were extracted with 0.4 Af ammonium sulfate. All solutions contained 1 mM PMSF (phenyl-methylsulfonyl fluoride). 

Preparation of pellet thaw the pellet in 37°C water. To the lysate, add 4 mL/g wet weight of 20 mM Tris-HCl, 40 mM magnesium acetate, 0.5% Triton X-100, pH 7.4 Add 30 U/g of DNase I and incubate for 30 min at ambient temperature with shaking Place a plastic pipet tip in the solution, cap, and sonicate 20 min in a bath sonicator (e g, Branson 1200) (see Note 2). Centrifuge this reduced viscosity sample at 15,000g for 45 min. The resulting supernatant contains CRABPI... 

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