With DEPC

DEPC-treated PBS (lOX, 3X, IX) for IL DEPC-treated lOX PBS 23.5 g Na2HP04,4.5 g NaH2P04, 87.6 g NaCl, 0.8 mL DEPC solution, 1000 mL dH20 autoclave for 20 min. Dilute with DEPC treated-dH20 to make 3X and IX. UV-irradiated double-distilled water (UV-ddH20) (see Note 5). 

Wash the cell pellet 3X with DEPC-treated IX PBS. 

These tissne sections are floated in a nuclease-free clean waterbath filled with DEPC-treated water. 

Graded alcohols Dilute 100% alcohol with DEPC-treated water to make 50, 70, and 95% alcohols. 

Wash the slides twice with DEPC-treated IX PBS and dehydrate in graded etha-nols 50, 70, 95%, and absolute ethanol, for 2 min each. 

Place them in a slide holder and treat with DEPC-treated IX PBS for 5 min. Dehydrate in graded alcohol 50, 70, 95, and 100% for 2 min each. 

In all procedures prior to hybridization, utmost precautions must be taken to prevent the contamination of ribonucleases, which results in the degradation of cellular RNA. Gloves must be worn throughout the steps. PBS and water must be autoclaved and, if possible, should be treated with DEPC (trrNote 4). All containers must be clean and ribonuclease-free. Particularly, it is important that, if reused, 12-well plates and insert buckets are treated with HjOj and DEPC (rrrNote 5). 

The phenol used for RNA extraction should be of the highest purity (double distilled), as oxidation products of phenol can cause degradation of RNA. Before use, the phenol is saturated with DEPC-treated water, and the phases are allowed to separate. The pH of the top aqueous layer is tested with pH paper. If phenol is acidic, it is equilibrated with Tris buffer by mixing it with a volume of 1 M Tris-Cl, pH 7.0. After phase separation, the top buffer layer is removed and the phenol is equilibrated twice with water. Additives such as 8-hydroxyquinoline (0.1%) are used in phenol to inhibit the activity of nucleases. Caution should be exercised in the use of phenol, as it is corrosive and a suspected carcinogen. Use in a chemical hood is recommended. Phenol chloroform extraction should be carried out in con-... 

Prepare reaction plate by placing 0.6 pL of 10% DMSO in DEPC dH20 or 0.6 pL of test compounds dissolved in 10% DMSO in DEPC dH20 per well. Four wells with DEPC dH20 should serve as controls, two for t = 0 and two for 1 = 30 min. Cover the plate with clear plastic packing tape and keep on ice. 

Water treated with DEPC as RNase-free water. 

DEPC overnight at room temperature, followed by autoclaving for 30 min at 120°C, to remove any traces of DEPC. Tris buffers may not be treated with DEPC. For their preparation, use a dedicated Tris container, RNase-free weighing materials, and DEPC-treated water. Before and after extraction, wipe all working surfaces with denatured ethanol or 10% bleach or, for nonmetallic surfaces, with RNase AWAY. 

Silica suspension, prepared as follows to a 2-L measuring cylinder containing 100 mL DEPC-water, add 60 g Si02 and fill up to 500 mL with DEPC-water. Stir on a magnetic stirrer until all the silica is resuspended. Leave suspension overnight at room temperature, to allow the silica to sink to the bottom of the... 

All solutions used for reverse transcription must be free of RNase. If all solutions are prepared with RNase-free water in disposable plastics and no contact with RNase-contaminated spatula or pH probes has occurred, the solutions need not be treated any further. Otherwise, to decontaminate the solutions add diethyl pyrocarbonate (DEPC) (e.g., Sigma, St. Louis, MO) to the solution to a final concentration of 0.1%, shake, let sit overnight with loosened caps, and then autoclave for 15 min. Note that DEPC might be carcinogenic and that solutions containing Tris cannot be decontaminated with DEPC, but must be prepared with special caution to prevent any RNase contamination. 

DEPC serves as a coupling agent for the synthesis of simple amides, esters, a-aminonitriles, and aryl thiocyanates it is also used as an efficient coupling agent for racemization-free peptide synthesis. Amides of various types can be obtained by the simple mixing of carboxylic acids and amines with DEPC in the presence of triethylamine. Both aromatic and aliphatic acids easily react with aromatic and aliphatic... 

Glassware used for RNA work should be cleaned with a detergent, thoroughly rinsed, and oven baked at 240 °C overnight or at 300 °C before use. Autoclaving alone does not fully inactivate many RNases. Alternatively, glassware can be treated with DEPC (diethylpyrocarbonate) as follows. 

As DEPC reacts vtith primary amines, it cannot be used directly to treat Tris buffers. When preparing Tris buffers, treat water with DEPC first, and then dissolve Tris to make the appropriate buffer. 

As in any procedure involving the handling of RNA it is vital to ensure that all equipment and solutions used are treated to remove ribo-nuclease activity and tiiat gloves are worn at all times to prevent contamination of the preparations with ribonucleases present in perspiration. Glassware should be siliconized, soaked in 0.2% DEPC for 1 h and then baked at 180°C for 2 h. Solutions, with the exception of Tris-HCl solutions and organic solvents, should be DEPC-treated prior to autoclaving see Note 1). Tris-HCl solutions should be prepared with DEPC-treated and autoclaved water. All chemicals used should be AnalaR grade,... 

Diethyl pyrocarbonate (DEPC) is a potent inhibitor of RNases see Note 1) and is used to treat all solutions except those containing Tris. Tris solutions should be made from a reserved stock of Tris crystals with DEPC-treated sterile distilled water in suitable containers and autoclaved before use. 

Plastic 1-mL syringes plugged with DEPC-treated glass wool. 

The homogenizer used should be kept as clean as possible. It should be washed extensively with DEPC-treated distilled water prior to use, between each sample, and upon completion of a batch of preparations. 

X SSC 3Af NaCl, 0.3M trisodium citrate, pH 4.5. Adjust pH with 1. OAf citric acid. Treat with DEPC before using. 

The fluorescence emission peak at 680nm decreased with DEPC... 

The chlorophyll fluorescence emission peak decreased 10 times with NBD-Cl (1000 pM) and about 3 times with DEPC (1000 pM). Both Tyr and His residues may be involved with the chlorophyll antenna as... 

MnClj typically inhibits DPC supported DCIP photoreduction of PS II membranes by 50% under the conditions of our assays (Fig. 1). Modification of available histidine residues with the modifying agent DEPC reduces the inhibition by MnClj to 25% [4,5]. Modification of carboxyl residues with EDC results in a similar reduction. Sequential modification of residues, first with DEPC and then with EDC results in MnCl2 being unable to inhibit DPC -> DCIP activity. Half of the high-affinity Mn-binding site is susceptible to DEPC and the other half to EDC. 

Figure 1. M11CI2 inhibition of DPC -> DCIP activity of Tris-treated PS II membranes (control, O). These membranes were then treated with DEPC (A), EDC ( ), or DEPC and EDC (O).

Rinse the required number of SW50.1 centrifuge tubes with DEPC-treated water and dry. Add 3.6mL of CsCl solution to each tube. Draw lysate through a 23-gage needle several times to shear genomic DNA, and carefully layer on top of the CsCl cushion. 

The GuSCN layer is aspirated down to and including 0.5 mL of the cushion. The tube is carefully filled with DEPC containing H O, allowed to stand 2-5 min, and then the water is aspirated. This is repeated twice. 

Suitable rocking microtome, disposable blades and holder (reduces chance of cxmtami-nation with RNase frcmi a generally used blade), bath fw secticms (well-cleaned with detergent, rinsed well with distilled water, and filled with DEPC water), and hot plate. 

Replace saline-ethanol with 70% (v/v) ethanol (prepared with DEPC-treated water), and incubate for 30 min (small embryos) or Ih Qarger material) on ice. 

Add 50pL 5M ammonium acetate (prepared with DEPC water) and 200 pL ethanol. Incnbate on ice for 30 min and spin in a microfuge for 15 min. 

Meanwhile, fill two troughs with DEPC water and place in the fume hood. To the second (acetylation bath), add 6mL triethanolamine and 1 mL acetic anhydride and mix with a small stir bar on a stirrer. 

Bring the mixture to a total volume of 50 fil with DEPC water. 

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