Recovery nucleic acid

A technical challenge with this step is to achieve RNA extraction of uniform quality and efficiency for each fraction. This is because the amount of RNA in each sucrose gradient fraction varies considerably and the high concentration of sucrose in the bottom fractions interferes with phase separation in typical phenol-based extraction steps. To address these problems, we spike each fraction with an aliquot of a foreign (control) RNA, which can be used later to correct for differences in RNA recovery (and reverse transcription efficiency) between samples. We then remove sucrose from the samples by precipitation of total nucleic acid and protein with ethanol. To purify RNA, a standard Trizol (Invitrogen) extraction is performed as outlined later (also see product insert). 

Pentynoic acid, 5 34t People. See also Personnel investment in, 21 624 organizational ties to, 21 627-628 People s Republic of China. See also China demand for oil in, 23 530 oil recovery program in, 23 534 Pepper, pipeline levels in, 23 159 Peptide antibiotics, 18 252-253. See also Antimicrobial peptides Peptide backbone hydrogen bonds, in proteins, 20 826 Peptide mapping, 3 840-841 Peptide nucleic acids, 17 631-634... 

Paul A. K., Martin D. A., and Robert D. P. (1990) Linearization of baculovirus DNA enhances the recovery of recombinant virus expression vectors. Nucleic Acids Kes. 18, 5667-5672. 

Eltoum, I., Fredenburgh, J., and Grizzle, W. E. 2001. Advanced concepts in fixation. 1. Effects of fixation on immunohistochemistry, reversibility of fixation and recovery of proteins, nucleic acids, and other molecules from fixed and processed tissues. 2. Developmental methods of fixation. J. Histotechnol. 24 201-210. 

Jurinke C, van den Boom D, Collazo V, Luchow A, Jacob A, Koster H. Recovery of nucleic acids from immobilized biotin-streptavidin complexes using ammonium hydroxide and applications in MALDI-TOF mass spectrometry. Anal Chem 1997 69 904-910. 

The methods used in the recovery, extraction, and purification must be described in detail. Special attention must be paid to the elimination of viruses, nucleic acids, and undesirable antigenic materials. 

This chapter will focus on a unique problem encountered during recovery of intracellulary produced proteins. Disruption of cells produces a mixture of nucleic acids and proteins in the solution from which the desired proteins must be fractionated. Typical separation schemes involve first the removal of nucleic acids from solution by precipitation. The desired protein is then isolated and purified from the mixture of remaining nucleic acids and proteins. A scheme for recovery of intracellular bacterial enzyme tartrate dehydrogenase from cell paste is shown in Fig. 1. Material balance at the different stages of the scheme in two different experiments showed that 53-60% loss in enzyme activity took place during precipitation of nucleic acids by protamine sulfate and during ammonium sulfate fractionation of proteins (Table 2). Reduction in volume, removal of major nonprotein... 

With an extract of E. coli, the effectiveness in precipitating nucleic acids decreased in the order polylysine > polyethyleneimine > cetavlon > streptomycin sulfate > protamine sulfate > MnCl2 > spermine. With MnCl2, precipitation was relatively inefficient and as much as 75% nucleic acids remained in solution. Extracts of Micrococcus lysodiekticus treated with protamine sulfate showed a loss of catalase activity and ineffective removal of RNA. In contract, treatment of a similar extract with polyethyleneimine resulted in 90% precipitation of nucleic acids and 70% recovery of catalase... 

Melling and Atkinson29 investigated nuclease treatment as a method for the removal of nucleic acids from bacterial suspensions. Two strains of E. coli were used and for both the strains the nuclease treatment was effective in depolymerizing nucleic acids and, hence, in recovery of supernatant after centrifugation to remove cell debris from disrupted cells. The nucleotide content in the supernatant was found to be 15-20% of total proteins and nucleic acids. The nucleotide content in the supernatant was reduced to a very low level by ammonium sulfate precipitation followed by dialysis of the redissolved precipitate. As stated earlier, direct precipitation of nucleotides resulted in significant residual nucleotides in the proteins. 

In conjunction with research on protein extraction from yeast, we investigated methods for the maximum recovery of protein possessing good functional properties but low in nucleic acid. Therefore, we examined the feasibility of making the yeast protein resistant to proteolysis during extraction and nucleic acid reduction. Using established extraction procedures (76), we observed... 

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