Isolating RNA is easy and fun.

Isolating RNA is easy and fun. This regular paper is available in Fungal Genetics Reports: https://newprairiepress.org/fgr/vol33/iss1/2 Akins, R.A., A.R. Kubelik and We reported previously the development of a general method for cloning Neurospora nuclear genes A.M. Lambowitz by sib selection, using a library of N. crassa genomic DNA fragments in plasmid pRAL1 (Akins Map of Plasmid pRAL1 and Lambowitz Mol. Cell. Biol. 5:2272-2278, 1985). Fig. 1 is a revised map of plasmid pRAL1. The size of the plasmid is measured more accurately as 4.7 kb, rather than 4.4 kb reported previously. In addition, the position of the EcoR1 site in the qa-2^+ gene was indicated incorrectly in the previous map. The sizes of the EcoR1 fragments are 2.8 and 1.85 kb. We and others have now cloned at least ten genes using the pRAL1 library: nic-1 and inl (Akins and Lambowitz, Molec. Cell. Biol. 5:2272-2278, 1985), cyt-18 (Akins and Lambowitz, unpubl.), cyt-(297-24) Kuiper, de Vries, Akins and Lambowitz, unpubl.), cyt-4 (Serizawa, Akins and Lambowitz, unpubl.), cyt-(289-4) (Kubelik and Lambowitz, unpubl.), his-2 (Akins, Lambowitz and Kinsey, unpubl.), van (Mann, Metzenberg, Akins and Lambowitz, unpubl.), cys-3 (Paietta, Marzluf, Akins and Lambowitz, unpubl.) and met-7 (Dr. M. Case, University of Georgia, personal communication). The library is available to all investigators. This work supported by NIH grant GM23961. Dept. of Biochemistry, St. Louis University School of Medicine, St. Louis, MO 63104 Chambers, J.A.A. and V.E.A. Russo We have found that the common phenol or guanidium thiocyanate based procedures for isoIsolating RNA is easy and fun lating RNA simply do not repay the effort for the yields involved (0.2-0.3 mg total RNA per g fresh weight with guanidium salts). A number of observations led us to examine and slightly modify a procedure originally developed for tissue culture cells (Auffray and Rougeon, 1979, Eur. J. Biochem. 107:303). This somewhat unusual procedure is based upon lithium chloride/urea solubilization of the cellular contents and the ultimate precipitation of RNA at high ionic strength. The combination of effective solubilization, inhibition of RNAse activity, effective precipitation of RNA, and a minimum of handling results in higher yields (0.5-1 mg RNA per g fresh wt.) for much less effort.