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1.
为解决在一定时间内DNA提取野外采样的携带与运输问题,本研究就用于DNA提取的胡杨叶片的非低温保存方法进行了探索。研究结果表明,在选用的5种胡杨叶片非低温保存方法中,70%乙醇,加入50mmol/LEDTA的70%乙醇以及TE缓冲液的保存效果欠佳,保存4d时获得的DNA多降解成弥散片段;采用硅胶干燥保存和SDSDNA提取液保存方法处理的胡杨叶片,在保存20d时仍能获得完整性好、纯度高的DNA大片段。AFLP证明硅胶干燥保存和SDSDNA提取液保存的叶片可取得与新鲜叶片提取DNA相一致的实验效果,该两种方法可作为胡杨野外采样选用的保存方法。  相似文献   

2.
目的:找出适合DNA提取的昆虫标本保存方法。方法:用几种常用的昆虫标本保存方法对蜜蜂处理不同时间后,用蛋白酶K法对其基因组DNA进行提取和纯化,然后对提取产物做琼脂糖凝胶电泳及紫外吸收分析。结果:75%乙醇及冻存处理材料的基因组DNA得率较高,为7.13~8.85μg/g,电泳条带较亮;甲醛处理材料的基因组DNA得率较低,为1.50~3.21μg/g。结论:用75%乙醇及冻存处理蜜蜂较适合于其基因组DNA的提取,不宜用甲醛。  相似文献   

3.
通过比较不同保存方法对柽柳(Tamarix chinensis)基因组总DNA提取效果的影响,确定合适的野外采集植物样品的保存方法。采用改良CTAB法提取了室温风干、4℃、-20℃、-80℃和硅胶干燥5种保存方法分别保存24 h、1周和1月后的柽柳基因组总DNA,结果表明:4℃保存1月后DNA产量最低,为60.35μg/g,-80℃条件下保存24 h的材料中提取的DNA产量最高,为246.92μg/g,保存效果最好。但在野外仪器条件不具备的情况下,柽柳可采用室温风干或硅胶干燥保存的方法,1月内材料中DNA降解程度较轻,可以扩增出所需目的片段。  相似文献   

4.
福尔马林保存的动物标本基因组DNA的提取方法   总被引:26,自引:0,他引:26  
从在福尔马林长期保存的动物标本中提取基因组DNA用于分子生物学研究一直是一个未得到彻底解决的难题。本文提出了一种从浸泡在福尔马林的大仓鼠肝脏和日本鳗鲡肌肉标本中提取基因组DNA的新方法。取浸泡于福尔马林中的大仓鼠肝脏或日本鳗2鲡肌肉适量,用PBS溶液冲洗,放在灭菌的吸水纸上将其揩干,于超净工作台内用无菌剪刀将材料剪成50mg的小块,放入PBS液浸泡12-24h;然后转入70%的乙醇中处理12-24h。依次换入下列梯度酒精中处理:80%乙醇,2h,重复一次;90%乙醇,2h,重复一次;95%乙醇,2h,重复一次;100%乙醇,1h,重复一次。然后将材料放入1/2倍的PBS液中浸泡12h。其间更换一次溶液。提取方法参考Sambroock等人(1989),加蛋白K瓣量按标准量(100μg/mlL),在50-56℃温浴3-6h处理过程中,不断轻摇混匀,视消化效果可以重复加入标准量的1/2倍蛋白酶K进行消化,直至将材料完全消化为止;酚氯仿抽提最后一次的上清液移入透析袋中透析;沉淀DNA时,在-20℃下20min效果为宜。该方法主要特点在于对标本进行预处理,在保证不使DNA进一步了解的前提下,首先去除标本中所含的福尔马林溶液的成分,然后利用改进的酚氯仿抽提法提取该类标本的基因组DNA,再进一步用透析法纯化DNA。研究结果表明,采用该方法提取和纯化被试标本基因组DNA能较好地应用于RAPD,微卫星位点的PCR扩增、Suthern和斑点杂交。  相似文献   

5.
不同保存方式下蝗虫组织DNA的提取及RAPD分析   总被引:17,自引:0,他引:17  
为了开展蝗虫分子系统学研究,分别对冷冻、乙醇浸泡(100%、乙醇、70%乙醇)和干制蝗虫标本用饱和NaCl法进行了基因组DNA的提取,并用随机引物进行扩增,结果表明:70%乙醇固定的标本和部分干标本提取的总DNA得率较低,在琼脂糖凝胶电泳检测中大音琏分有明显降解,导致PCR扩增中信息缺失,甚至无扩增条带;而保存完好的干标本、-20℃冷冻标本和100%,乙醇浸泡标本提取的总DNA带型整齐,无拖尾,PCR扩增结果的稳定性好,成为蝗虫分子系统学研究中首选的三种保存方式。  相似文献   

6.
几种提取枣和酸枣DNA用于RAPD分析的方法比较   总被引:12,自引:0,他引:12  
利用不同的方法提取枣和酸枣的总DNA,并分析了不同样品状况、抗氧化剂和纯化过程等对所提DNA质量及其RAPD扩增效果的影响。实验显示:用改良CTAB法优于SDS法,可有效去除多糖。加入的抗氧化剂PVP、抗坏血酸、β-巯基乙醇等可有效阻止多酚类物质褐化,但不同抗氧化剂间效果差别不大。分别将样品干燥处理、固定处理、液氮处理、冷藏,与新鲜材料相比,用液氮处理并保存于-80℃的材料与新鲜材料所提DNA相当,而用其他方法DNA都有不同程度降解。不同材料提取结果比较显示:幼叶所提DNA产量和质量优于老叶,并且不需要较多的纯化过程。在有RNA和少量蛋白质时,对扩增结果影响不大。  相似文献   

7.
黑麂粪便DNA提取及其PCR检测   总被引:6,自引:1,他引:5  
采集了黑麂(Muntiacuscrinifrons)的新鲜粪便以及在野外自然条件下保存较长时间的粪便样品,晾干后带回实验室,提取其DNA;同时提取黑麂肌肉、皮张样品的DNA,用以对比粪便样品的提取效果。电泳检测结果显示,此方法使用实验室中常用的分子生物学试剂,可以从黑麂粪便样品中抽提到高质量的粪便DNA并克服分子粪便学研究中常见的PCR反应抑制物的影响。为其它濒危鹿科动物的非损伤性取样提供了的新途径,为其遗传结构、遗传多样性现状等研究提供了更加广阔的取材空间。  相似文献   

8.
高质量甘蔗基因组DNA的简便快速提取方法研究   总被引:4,自引:0,他引:4  
甘蔗是世界上重要的糖料作物和能源作物。目前,甘蔗分子生物学研究已成为甘蔗研究的热点之一。基因组DNA的提取是进行甘蔗分子生物学研究的基础。本研究设计含一系列SDS浓度的提取液,同时设加液氮和不加液氮研磨的对比试验,提取甘蔗不同部位叶片的基因组DNA并进行产量和纯度检测以及分子生物学分析。结果表明,所有提取液提取的甘蔗基因组DNA纯度均很高,A260/A280在1.8-2.0之间,A260/A230大于2,但提取液I(0.75%SDS)提取的甘蔗基因组DNA产量较低;加液氮与否对甘蔗基因组DNA的提取产量和纯度没有影响;以提取的甘蔗基因组DNA为模板,分别用一对扩增SPS(蔗糖磷酸合成酶)基因部分片段的引物和一对ISSR引物进行PCR扩增,所有DNA均能扩增出预期的条带;用不同的限制性内切酶对所提取的甘蔗基因组DNA进行酶切,所有DNA样品均能完全酶切。本研究得出最佳甘蔗基因组DNA提取方法如下:磨碎甘蔗叶片后,加DNA提取液(SDS:1.5%;Tris:100 mM;EDTA:20 mM;NaCl:500 mM)于65℃裂解30 min,经酚∶氯仿和氯仿各抽提一次,可获得高产量高质量的甘蔗基因组DNA,能满足后续分子生物学研究的要求。  相似文献   

9.
基因组和转录组的高通量测序对样品的纯度和含量要求较高,旨在通过研究高山被孢霉在不同培养条件下和采用不同菌丝处理方法对其基因组和转录组质量的影响来确定最佳提取方式。结果表明,PDA培养基培养的菌丝与发酵培养基培养的菌丝经基因组提取后在琼脂糖凝胶电泳中有明显不同的表现,使用发酵培养基培养的菌丝提取的基因组DNA无降解,而以PDA培养基培养的菌丝提取的基因组DNA出现部分降解。在使用国产试剂盒做高山被孢霉转录组RNA提取实验中发现,使用液氮速冻后破碎菌丝并结合使用β-疏基乙醇更易提取到高浓度且无降解的转录组样品。  相似文献   

10.
目的比较两种肠内容物前处理和两种提取方法对清洁级SD大鼠肠内容物细菌基因组DNA提取效率。方法分别选用PBS多次离心漂洗、液氮破细胞两种前处理方法和酚/氯仿抽提、试剂盒过柱法两种提取方法进行组合分析,对4份肠内容物和16份含金黄色葡萄球菌肠内容物进行随机提取。结果大鼠肠内容物细菌基因组DNA含量和纯度测定结果显示,与PBS反复离心相比,液氮研磨前处理能显著提高大鼠肠内容物基因组DNA。荧光定量PCR表明,液氮研磨前处理较PBS反复离心能更好地收集细菌基因组DNA,其Ct值最低。结论研究结果表明,采用液氮研磨试剂盒法在大鼠肠内容物DNA提取中是较为优良的方法,该方法为建立实验动物中微生物的定量PCR检测方法打下了基础。  相似文献   

11.
The occurrence of chironomids in a standard pond net sample was studied. The standard pond net sample is quite biased; its qualitative to semi-quantitive character should always be taken into account. It is advised to transform abundances (log-normal) of chironomids when collected with the standard pond net. Despite disadvantages the pond net can be used in water quality surveys because of their general objectives.  相似文献   

12.
Precision of the estimate of the population mean using ranked set sample (RSS) relative to using simple random sample (SRS), with the same number of quantified units, depends upon the population and success in ranking. In practice, even ranking a sample of moderate size and observing the ith ranked unit (other than the extremes) is a difficult task. Therefore, in this paper we introduce a variety of extreme ranked set sample (ERSSs) to estimate the population mean. ERSSs is more practical than the ordinary ranked set sampling, since in case of even sample size we need to identify successfully only the first and/or the last ordered unit or in case of odd sample size the median unit. We show that ERSSs gives an unbiased estimate of the population mean in case of symmetric populations and it is more efficient than SRS, using the same number of quantified units. Example using real data is given. Also, parametric examples are given.  相似文献   

13.
Biological materials obtained during routine veterinary treatment and necropsies on zoo and aquarium animals provide opportunities to study a wide variety of phenomena, many of which have important implications for wildlife conservation and captive animal management. Collection and shipment of biological materials require extensive coordination, and few zoological institutions have developed detailed, standardized protocols to facilitate these efforts. The value of using biological materials is reviewed and a set of policies and guidelines are outlined for their collection, preservation, and shipment. Several related issues are discussed, including health hazards and liability issues, permit requirements, and animal welfare considerations.  相似文献   

14.
Terrestrial tardigrades are often found in the lichens and mosses growing on trees and rocks. The assertion that tardigrades in these habitats are very patchy in their distribution has rarely been backed by quantitative sampling. This study assesses spatial variability in tardigrade populations inhabiting small patches (0.1 cm2 to over 5 cm2) of moss and lichen on trees and rocks at three sites in the United States of America. Tardigrades were collected from four replicate rocks in the Ouachita Mountains of Arkansas, with 30 lichen patches collected on two adjacent boulders and 20 moss patches on a second pair of boulders. In Fort Myers and in Citrus Springs, Florida, 30 lichen patches per tree were collected from two pairs of trees. The tardigrades in each sample were extracted, mounted, identified, and counted. The variation in tardigrade abundance among lichen or moss patches within rocks or trees was very high; the only consistent pattern was that very small patches usually lacked tardigrades. Tardigrade diversity and abundance also varied greatly within sites when lichens and mosses of the same species from different rocks and trees were compared (in the most extreme case one tree had numerous individuals of two tardigrade species present while the other had almost no tardigrades). The results of this quantitative sampling support the assertion that tardigrades are very patchy in distribution. Given the considerable time investment required for the quantitative processing of tardigrade samples, this high spatial variability in tardigrade diversity and abundance requires that researches testing ecological hypotheses about tardigrade abundance check variability before deciding how many samples to take.  相似文献   

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16.
吉林蛟河42 hm2针阔混交林样地植物种-面积关系   总被引:1,自引:0,他引:1       下载免费PDF全文
 种-面积关系是生态学中的基本问题, 其构建方式对种-面积关系的影响以及最优种-面积模型的选择仍然存在争议。该文利用吉林蛟河42 hm2针阔混交林样地数据, 分别采用巢式样方法和随机样方法建立对数模型、幂函数模型和逻辑斯蒂克模型, 并通过赤池信息量准则(AIC)检验种-面积模型优度。结果表明, 种-面积关系受到取样方法的影响, 随机样方法的拟合效果优于巢式样方法。采用随机样方法构建的幂指数模型(AIC = 89.11)和逻辑斯蒂克模型(AIC = 71.21)优于对数模型(AIC = 113.81)。根据AIC值可知, 随机样方法构建的逻辑斯蒂克模型是拟合42 hm2针阔混交林样地种-面积关系的最优模型。该研究表明: 在分析种-面积关系时不仅应考虑尺度效应, 还需注意生境变化及群落演替的影响。  相似文献   

17.
The ABI Sequencing Analysis application is designed specifically for the analysis of data produced by the ABI DNA Sequencer. The ABI sequencer is a laser-based instrument that utilizes fluorescent labels to analyze the products of a sequencing reaction as they migrate through a gel. After the data are collected from a sequencing run, the Analysis program identifies and tracks the sample lanes of the gel and subsequently normalizes and integrates the raw data into a chromatogram of the final sequence. For the use, there are basically two types of files that can be manipulated to potentially improve the analysis results. The Gel File consists of a computer generated image of the sequencing gel with the fluorescent DNA banding patterns. This image allows the user to view and edit the tracking lines generated and used by Analysis to collect data points for each sample. Individual Sample Files are stored for each of the samples analyzed and include the chromatogram, raw data, and annotations and information regarding the sample and sequence run. Generally, the products of a sequencing reaction are easily resolved and the Analysis software interprets the correct nucleotide sequence. Ambiguous base calls tend to occur near the end of the sequence and may be either edited or deleted by the user before exporting the data for further comparisons or alignments. Occasionally the tracking lines within the gel image may need to be adjusted or moved. The sample data are then reextracted from the Gel File and analyzed again. This review explains the general operation of Analysis in terms of viewing and editing a chromatogram, retracking the lanes of a Gel File, and analyzing the final sample data. The three versions 1.2.1, 2.1.2, and 3.3 are discussed.  相似文献   

18.
Despite recent developments in bottom‐up proteomics, the need still exists in a fast, uncomplicated, and robust method for comprehensive sample processing especially when applied to low protein amounts. The suspension trapping method combines the advantage of efficient SDS‐based protein extraction with rapid detergent removal, reactor‐type protein digestion, and peptide cleanup. Proteins are solubilized in SDS. The sample is acidified and introduced into the suspension trapping tip incorporating the depth filter and hydrophobic compartments, filled with the neutral pH methanolic solution. The instantly formed fine protein suspension is trapped in the depth filter stack—this crucial step is aimed at separating the particulate matter in space. SDS and other contaminants are removed in the flow‐through, and a protease is introduced. Following the digestion, the peptides are cleaned up using the tip's hydrophobic part. The methodology allows processing of protein loads down to the low microgram/submicrogram levels. The detergent removal takes about 5 min, whereas the tryptic proteolysis of a cellular lysate is complete in as little as 30 min. We have successfully utilized the method for analysis of cellular lysates, enriched membrane preparations, and immunoprecipitates. We expect that due to its robustness and simplicity, the method will become an essential proteomics tool.  相似文献   

19.
A method of analysis for the determination of alosetron in human plasma or serum has been developed. The method was fully automated using a laboratory robot in order to improve analytical precision, efficiency and safety. The assay involved solid-phase extraction with reversed-phase HPLC separation and fluorescence detection. A validation exercise over the concentration range of 0.1 to 20 ng/ml demonstrated the selectivity, linearity, sensitivity, accuracy, precision, extraction efficiency, ruggedness and stability of the method. The method has been applied in support of numerous human pharmacokinetic/biopharmaceutic studies over the last five years.  相似文献   

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