By RALPH RAPLEY
Leading edge organic investigators supply a robust and hugely necessary choice of updated tools for the isolation of RNA from various resources, together with bacterial, plant, and mammalian cells. those fine-tuned protocols take either the skilled and green investigator in the course of the nuances of RNA manipulation, from extraction to in vitro translation. The booklet additionally comprises protocols for the research and characterization of remoted RNA species and broad troubleshooting tricks and how one can be sure effortless reproducibility. RNA Isolation and Characterization Protocols is the only top number of simply reproducible, step by step RNA concepts now to be had for brand new bench scientists.
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Example text
The upper, PEG-rich phase will occupy some 80% of the total volume and the salt-rich, lower phase 20%, but the latter contains about 80% of the total RNA 3. Some organisms (P aeruginosa, K. aerogenes) may be extracted under the conditions given, whereas others that have been tried (Salmonella typyhimurzum, Proteus mwabrlis, Serratia marcescens) will require changes in some concentratrons of reagents, in particular SDS and/or PEG may be increased 4. If the RNA IS not to be precipitated and washed, and should small amounts of phenol interfere with subsequent treatment of the RNA, then phenol can be removed by washing with chloroform Add about an equal volume of chloroform, mix and centrifuge very brrefly, discard the lower, chloroform layer.
Notes 1. Since guanidimum thiocyanate is hazardous, it IS best to prepare this solution m the manufacturer’s bottle without weighing to mmlmlze handlmg. When making a smaller quantity of the solution, wear gloves when weighing. This solution can be stored at least 3 mo at room temperature. 2. To mmimlze handling, dissolve 500 g crystal phenol in the manufacturer’s bottle with 500 mL sterile deionized water Store in 50-rnL aliquots m a -20°C freezer For routme use, this solution can be kept at 4°C for up to 1 mo.
And Kootstra, A. (1993) Isolation of RNA from apple skin. Plant Mol. Blol Reptr 11, 326-332 3. Newbury, H. J. and Possmgham, J. V. (1977). Factors affecting the extraction of mtact ribonucleic acid from plant tissues contaming interfering phenolic compounds. Plant Physiol. 60,543-547. 4. Schultz, D. , Cox-Foster, D. , Mumma, R. , and Medford, J I. (1994) RNA isolation from recalcitrant plant tissue. Plant Mol Biol. Reptr. 12,3 10-3 16. 5. -S. and Vodkin L. 0. (1994) Extraction of RNA from tissues containing high levels of procyamdms that bmd RNA.