Ribonuclease avoidance

In order to obtain large amount of proteins with this wheat germ cell-free protein synthesis system, even trace contaminations of ribonucleases should be avoided throughout the transcription and translation steps. Therefore, great care must be... 

The use of buffer solutions is usually advisable for accurate control of the pH of solutions used in spectrophotometric titrations, since the solutions will usually be so dilute (because of the relatively high protein or peptide absorptivity) as to offer little self-buffering. The usual buffers for the pH range from 9 to 13, i.e., borate, glycinate, phosphate, lysine, -aminocaproic acid, etc., are generally transparent through the 2950 A phenolate band. Buffer systems composed of piperidine (pK, 11) and its hydrochloride have been used to avoid the use of multivalent anions in the spectrophotometric titration of ribonuclease (Klee and Richards, 1957), but no other advantage is apparent for this system. 

To probe for one-dimensional diffusion, we synthesized DNA/RNA chimeric oligonucleotides. Special precautions were taken to avoid ribonuclease contamination during synthesis, purification, and use of these chimeras. For example, all water was treated with diethylpyrocarbonate before exposure to the chimeras. Ribonucleotide 2 -hydroxyl groups were deprotected with 1 M tetrabutyl ammonium fluoride in dimethyl formamide (Aldrich Chemical Milwaukee, WI). Purified oligonucleotides were labeled on the 5 end with [y-32p]ATP (duPont Wilmington, DE) by T4 kinase (Promega Madison, WI), and desalted with a Nick gel filtration column (Pharmacia Uppsala, Sweden). 

Need to verify that radioactivity is not due to contamination or metaboUsm, i.e., to avoid possible artifacts, HPLC of DNA hydrolysates and digestion with proteases and ribonucleases can be used. 

More recently, EMC virus RNA was translated in a rabbit reticulocyte lysate made mRNA-dependent by prior treatment with micrococcal nuclease (1 )- This system has been shown to maintain a high elongation rate, to be relatively free of ribonuclease activity, and to avoid premature termination on other mRNAs (I8). 

Care should be taken to avoid the presence of residual detergents on beakers, measuring cylinders, etc., as this will compromise the extract. All centrifuge tubes, pipette tips, and other materials likely to come into contact with the extract after lysis should be autoclaved to inactivate ribonuclease activity. 

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