Preparing Neurospora DNA: some improvements.
Preparing Neurospora DNA: some improvements.
复制标题
制备脉孢菌 DNA：一些改进。
DOI：
10.4148/1941-4765.1643
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发表时间：
1982
期刊：
影响因子：
--
通讯作者：
R. L. Metzenberg
中科院分区：
文献类型：
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作者：
J. N. Stevens;R. L. Metzenberg
神经孢子虫DNA的制备：一些改进。本作品采用知识共享署名-相同方式共享4.0许可协议。该技术说明可在真菌遗传学报告中找到：http://newprairiepress.org/fgr/vol29/iss1/14 Selitrennikoff, c.p.神经孢子菌的原生质体可以通过使用一种“细胞壁酶”处理菌丝而形成。然而，对温度敏感、原生质体形成产生的原生质体往往有残余细胞壁和os-1变异的神经孢子虫的一些花招。不会成长和分裂。在一定条件下，神经孢子菌的黏液变种会以原生质体的形式生长。不幸的是，这样的文化不会参与杂交，从而使基因操作变得困难。为了克服这个和其他困难，我们使用了一种温度敏感的渗透突变体（os-1 (NM233t)）来形成原生质体。一般方法以前已经发表过（Selitrennikoff et al., 1981 Experimental Mycology 5: 155-161），这里包括一些细节。从库存中心可获得os-1 （NM233t）的菌丝分离株的两种交配型，并可作为雄性或雌性亲本，以构建任何所需的基因型。后代可以在琼脂斜坡上生长，温度为25°或37°C。将大分生孢子（1-10 × 105个细胞/ml）接种到含有50 ml以下培养基的250 ml烧瓶中：10% (w/v) Sorbose；2% （w/v）蔗糖；1 x Vogel's盐，400 μg/ml Polyoxin B. Polyoxin B是几丁质合成酶活性的抑制剂，从Polyoxin AL可湿性粉末中纯化而来（可在日本非处方购买，也可从Kaken Chemical Co., Ltd., 28-8 2-Chome, Honkomagome, Bunkyo-Ku, Tokyo, Japan）。将100克粉末溶解在1升水中，不溶性物质通过使用whatman# 1纸过滤去除。棕色滤液应用于Norite柱（颗粒状粉末包装到非常慢（100 ml/天）柱）。用清水冲洗色谱柱，用60%的丙酮洗脱多辛。脏棕色的馏分汇集在一起，丙酮被冲出来。所得到的棕色“粘稠物”或粉末溶解在水中，并应用于Dowex 50 （8X，H+形式）柱。用0.3N洗脱多毒素，所得溶液冷冻干燥两次（以确保完全去除NH4OH）。典型批次的产品纯度为70%，紫外吸收率为25.35%。多毒素粉末可在4°C（或-20°C）下干燥保存较长时间。培养物在37°C下摇晃（140 rpm）培养，每天通过无菌玻璃棉过滤成新鲜培养基。7天后，原生质体稳定培养。重要的是，在初始转移期间，细胞密度保持在5 × 105个细胞/ml或更高。然后将培养物过滤到含有不含多聚辛的培养基的烧瓶中，每天（过滤）转移到新鲜培养基中，再持续7天。然后，将它们过滤到培养基中，其中山梨糖已被7.5%的山梨糖醇取代，并允许其继续生长一周（当然，每天都要补充水分）。产生的原生质体群体在长达一年的生长中是稳定的，并且原生质体可以在温度转移到25°C时再生菌丝（包括大分生孢子）。然而，由于原因尚不清楚，所得到的菌丝体在形态上与原始的os-1菌株不同。原生质体可以在-70°C的培养基中保存至少两年。这些对温度敏感的原生质体在许多研究中非常有用，包括外源DNA的宿主（部分由美国国家科学基金会资助给CPS）。科罗拉多大学健康科学中心解剖学系，科罗拉多州丹佛80262。Stevens， J. N.和R. L. Metzenberg制备神经孢子菌DNA：一些改进。我们需要从相当多的不同菌株中制备适合于限制性内切酶分析的DNA，这使我们开发了一种简单的方法来制备DNA （Metzenberg和Baisch 1981 Neurospora Newsl. 28: 20-21）。从那时起，我们做了一些小的修改，结果在不增加努力的情况下显著提高了产品的产量和质量。(1)将“萃取缓冲液”用三乙基氨基乙二胺四乙酸酯（TEA.EDTA）取代EDTA锂，并加入适量的NaCl。这个过程的演变始于EDTA的钠盐，它是从乙醇中提取的难处理的糖浆。然后我们改用锂盐，它即使在高浓度下也不会“糖浆化”，但可能会在乙醇溶液中结晶，尤其是在实验室播种后。三乙基铵盐没有给我们带来任何麻烦，只是我们又绕了一个圈，必须添加少量的钠离子以确保DNA的沉淀！茶。将73 g （250 mmol）游离EDTA酸悬浮于350 ml水中，加入三乙胺碱，使pH值为8.0 - 8.2，制成500 mM的EDTA原液。（如果有明显的有色杂质，应重新蒸馏。）在我们手中，所需的碱的体积是108.5毫升，但毫无疑问，这是根据三乙胺的含水量而变化的。然后将原液稀释到500毫升。我们将其冷冻保存，尽管这可能不是必要的。因此，“萃取缓冲液”中的最终浓度为：50 mM；细菌蛋白酶250 μg/ml。茶。EDTA, 250 mM；Triton X-100, 0.5%；(2)在33°或37°温和搅拌下提取2-3天的粉末似乎比在没有任何搅拌的情况下过夜提取的产量更高。这似乎在大规模准备工作中尤其如此。(3)所有的沉淀，无论是用乙醇还是用高氯酸乙醇，都在室温下用试剂和制剂进行。没有必要在4°或室温下对乙醇悬浮液进行孵卵，以确保DNA沉淀的完整性。(4)我们没有通过离心收集沉淀的DNA，这种方法会将任何酒精不溶性杂质与DNA一起沉淀，我们立即使用超市或酒类商店出售的箭头头塑料鸡尾酒牙签收集纤维状的DNA。（将牙签浸在氢氧化钠中，然后再浸在无菌水中，就可以去除可能存在的dna酶的痕迹）。多余的酒液从牙签中排出。丢弃不含DNA的混浊悬浮液，在室温下将DNA溶解在0.8倍所需终体积的低盐缓冲液中。这通常需要不到30分钟；然后加入0.2体积的5倍高盐缓冲液，如果该过程要在该点停止超过一个小时，或者最终的制剂要储存。威斯康星大学生理化学系，威斯康星麦迪逊53706
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.