Preparing Neurospora DNA: some improvements.

Preparing Neurospora DNA: some improvements. Creative Commons License This work is licensed under a Creative Commons Attribution-Share Alike 4.0 License. This technical note is available in Fungal Genetics Reports: http://newprairiepress.org/fgr/vol29/iss1/14 Selitrennikoff, C. P. Protoplasts of Neurospora can be formed by treating hyphae with one of a number of "cell-wall'ases". However, Temperature-sensitive, protoplast-forming resulting protoplasts often have residual cell-wall and os-1 variant of Neurospora a few tricks. will not grow and divide. The slime variant of Neurospora will, under certain conditions, grow as a population of protoplasts. Unfortunately, such cultures will not participate in crosses thus making genetic manipulation difficult. In efforts to overcome this and other difficulties, we have used a temperature-sensitive osmotic mutant (os-1 (NM233t)) in order to form protoplasts. The general method has been previously published (Selitrennikoff et al., 1981 Experimental Mycology 5: 155-161) and included here are some details. Mycelial isolates of os-1 (NM233t) are available in either mating type from the Stock Center and can be used as a male or female parent in order to construct any desired genotype. Progeny can be grown on agar slants at either 25° or 37°C. Macroconidia are inoculated (1-10 x 105 cell/ml) into 250 ml flask containing 50 ml of the following medium: 10% (w/v) Sorbose; 2% (w/v) Sucrose; 1 x Vogel's salts, 400 μg/ml Polyoxin B. Polyoxin B is an inhibitor of chitin synthetase activity and is purified from Polyoxin AL Wettable Powder (available either overthe-counter in Japan or from the Kaken Chemical Co., Ltd., 28-8 2-Chome, Honkomagome, Bunkyo-Ku, Tokyo, Japan). One hundred grams of powder are dissolved in 1 liter of water and insoluble material is removed by filtration using Whatman #1 paper. The brown filtrate is applied to a Norite column (granular form the powder packs to a very slow (100 ml/day) column). The column is washed with water and the Polyoxin eluted with 60% aqueous acetone. The dirty-brown fractions are pooled and the acetone is flushed out. The resulting brown "goo" or powder is dissolved in water and applied to a Dowex 50 (8X,H+ form) column. NH4OH The Polyoxin is eluted with 0.3N and the resulting solution freeze-dried twice (to insure complete removal of NH4OH). Typical batches result in a product 70% pure based on U.V. absorption with yields of 25.35%. Polyoxin powder can be stored desiccated at 4°C (or -20°C) for long periods of time. Cultures are grown at 37°C with shaking (140 rpm) and are filtered daily through sterile glass wool into fresh medium. After seven days a stable culture of protoplasts results. It is important that during the initial transfers that the cell density be maintained at 5 x 105 cells/ml or greater. Cultures are then filtered into flasks containing medium lacking Polyoxin and transferred daily (with filtration) to fresh medium for another seven days. These, then, are filtered to medium in which Sorbose has been replaced by 7.5% Sorbitol and growth allowed to continue for yet another week (with, of course, daily fitration). The population of protoplasts that results is stable for up to one year of growth and protoplasts can regenerate a mycelium (including macroconidia) upon a temperature shift to 25°C. However, far reasons that are not clear, the resulting mycelium is different morpholocially from the original os-1 strain. Protoplasts may be stored at -70°C in their medium for at least two years. These temperature-sensitive protoplasts are very useful for a number of studies, including hosts for exogenous DNA's, (Supported in part by NSF grants to CPS.) Department of Anatomy, University of Colorado Health Sciences Center, Denver, Colorado 80262. Stevens, J. N. and R. L. Metzenberg Preparing Neurospora DNA: some improvements. Our need to prepare DNA suitable for restriction enzyme analysis from rather large numbers of different strains led us to develop an easy method for making DNA (Metzenberg and Baisch 1981 Neurospora Newsl. 28: 20-21). Since then, we have made some small modifications that result in significantly improved yield and quality of product without increase in effort. The changes are as follows: (1) "Extraction buffer" has been modified by substituting triethylamnonium ethylenediamine tetraacetate (TEA.EDTA) for lithium EDTA, and by adding a modest concentration of NaCl. The evolution of the procedure started with the sodium salt of EDTA, which comes out of ethanol as an intractable syrup. We then switched to the lithium salt, which does not "syrup out" even at high concentrations, but may crystallize out of ethanolic solutions, especially once the lab has been seeded. The triethylamnonium salt has given no trouble of any kind except that we have come full circle and must add a small amount of sodium ion to insure precipitation of the DNA! The TEA.EDTA is made as a 500 mM stock solution by suspending 73 g (250 mmoles) of the free acid of EDTA in 350 ml of water and adding, with continuous stirring, triethylamine base to give pH 8.0 8.2. (The base should be redistilled if colored impurities are evident.) In our hands, the volume of base required is 108.5 ml, but no doubt this will vary, depending on the water content of the triethylamine. The stock solution is then diluted to 500 ml. We have stored it frozen, though this may not be necessary. The final concentrations in "extraction buffer", then, are: 50 mM; and bacterial proteinase, 250 μg/ml. TEA.EDTA, 250 mM; Triton X-100, 0.5%; NaCl, (2) Extraction of the powder for 2-3 days at 33° or 37° with gentle agitation appears to give somewhat larger yields than overnight extraction without any agitation. This seems to be especially true of scaled-up preparations. (3) All precipitations, either with ethanol or ethanolic perchlorate, are done with reagents and the preparations at room temperature. No incubation of the ethanolic suspensions, either at 4° or at room temperature, is necessary to insure completeness of precipitation of DNA (4) Rather than collect the precipitated DNA by centrifugation, which sediments any alcohol-insoluble impurities along with the DNA, we immediately collect the fibrous mass of DNA with an arrowhead-tipped plastic cocktail toothpick such as are available at supermarkets or liquor stores. (The toothpicks are freed of possible traces of DNase by dipping them into IN NaOH and then into sterile water). Excess liquors are drained from the mass of the toothpick. The cloudy DNA-free suspension is discarded and the DNA is dissolved in the same tube in 0.8X the desired final volume of low-salt buffer at room temperature. This usually takes less than 30 minutes; then 0.2 volumes of 5X high salt buffer is added if the procedure is to be stopped at that point for more than an hour, or the final preparation is to be stored. Department of Physiological Chemistry, University of Wisconsin, Madison, Wisconsin 53706.