一种快速提取小麦基因组DNA的改良CTAB方法

旨在寻求一种微量、快速提取小麦DNA的方法。以小麦幼叶为材料,用改良CTAB法、改良SDS法、沸水浴法提取小麦叶片总DNA,通过琼脂糖凝胶电泳法、紫外吸收法和PCR检测DNA完整性和纯度。改良CTAB法提取小麦基因组DNA质量和纯度高、无降解,每人每天可以轻松提取200个DNA样品,对转基因植株的PCR检测显示扩增目标条带清晰一致,无假阳性,试验结果理想。改良SDS法所提DNA纯度和浓度虽略不如改良CTAB法,但仍然符合实验要求。沸水浴法提取量少且杂质多,PCR无有效扩增,结果不理想。改良CTAB法提供了一种简便、快速微量小麦DNA提取方法,适用于PCR和其他分子生物学研究。     

苦瓜基因组DNA的提取及ISSR扩增体系的优化

为了快速获取高质量的苦瓜基因组DNA,以便进行苦瓜白粉病抗性基因分子标记研究,比较了不同的DNA提取方法、不同部位的苦瓜叶片提取基因组DNA的产量和质量,探究了苦瓜基因组DNA提取的最佳方法和叶片的最适部位;研究了ISSR-PCR的退火温度,并采用正交设计法对影响ISSR-PCR的Mg²⁺、dNTPs、Taq酶、模板DNA以及引物浓度等5个因素进行了优化。结果表明,采用改良CTAB法从苦瓜顶端嫩叶中提取的基因组DNA OD260/OD280值在1.8~1.9之间,OD260/OD230值为2.0左右,对DNA样品进行琼脂糖凝胶电泳检测,主带清晰,降解较少,产量和纯度均较高,效果较好。优化后的ISSR-PCR反应体系为：2.5μL 10×PCR buffer,2.5 mmol/L MgCl₂,250μmol/L dNTPs,10 ng模板DNA,0.75 U Taq酶,引物浓度0.7μmol/L,反应总体积为25μL,引物UBC826最佳退火温度为53℃。该体系在多次重复中均能获得良好的扩增结果。苦瓜基因组DNA提取的最佳方法为改良CTAB法,最适合的部位为顶端嫩叶。     

果树基因组DNA的提取和PCR扩增测序方法

为避免多年生果树组织中的多糖和酚类物质影响基因组DNA的提取质量,在CTAB提取缓冲液中加入2%PVP-40和2% β-巯基乙醇,以改进的CTAB提取方法从苹果嫩叶中提取基因组DNA.所得DNA样品D260nm/D280nm比值为1.98～2.05,纯度高.当用DNA样品和设计的特异引物进行PCR扩增时,其PCR产物只出现一条清晰电泳谱带,质量满足测序要求,采用DNA纯化试剂盒纯化后可直接用于测序.该方法已成功用于"国光"和"金冠"苹果的基因组DNA测序,是一个操作简便、快捷和高效的方法,非常适用于果树基因组DNA的提取和测序.     

瘤胃微生物总DNA提取方法的比较

目前如何获得完整的瘤胃微生物总DNA是对瘤胃微生物在分子水平研究中关键的一步。瘤胃微生物总DNA分子片段很大,大概在15kb~20kb,因此需要选择一种合适的提取方法来获得完整的总DNA片段。笔者研究采用物理方法如玻璃珠研磨震荡法、反复冻融法、超声波破碎法、液氮研磨法等,化学方法如表面活性剂十二烷基磺酸钠（sodium dodecyl sulfate,SDS）、溴化十六烷三甲基铵（Cetyltrimethyl Ammonium Bromide,CTAB等）,酶解法（溶菌酶、蛋白酶K等）相结合,并对其进行比较,选择最佳的提取方法,使其符合以后PCR扩增要求以及其它后续研究工作。试验结果表明用玻璃珠研磨震荡加CTAB提取液的方法、反复冻融加SDS提取液的方法提取的瘤胃微生物总DNA的效果高于其它方法,并且通过电泳以及PCR检测,所提取的DNA的量适于进一步的分子生物学研究。     

玉米种子DNA快速提取及杂交种纯度的快速鉴定

本研究用玉米杂交种渝单8号及其双亲的干种子作为材料,使用NaOH溶液研磨玉米种子胚,提取基因组DNA,并利用多重PCR扩增进行玉米杂交种子的纯度鉴定.结果表明,利用玉米种子快速提取方法获得的DNA可进行正常的SSR扩增,并且从20对玉米核心引物中筛选出了8对扩增谱带呈双亲互补型的引物,将其中3对引物YISSR、phi072和phi299852组合起来扩增,扩增谱带清晰可辨,对渝单8号杂交种子的纯度进行准确鉴定.初步表明利用多重PCR技术鉴定玉米种子纯度是可行的,不仅简便快速,而且降低了检测成本.     

一种应用PCR缓冲液快速制备水稻DNA模板的方法

本研究介绍一种应用PCR缓冲液快速制备水稻DNA模板的方法。操作方法如下:取少量水稻叶片,用剪刀剪碎后移至200μL离心管或96孔PCR板中,在每个样品中加入50μL 0.5倍的PCR扩增缓冲液,置于PCR基因扩增仪上在95℃条件下煮12 min,取出,无需离心,所得提取液直接作为DNA模板用于PCR扩增。结果表明,该方法所制备的水稻DNA模板在PCR扩增中稳定、准确,其效果与用常规SDS法提取的相当,且这种DNA模板可在短期内保存而不影响扩增效率。该方法具有成本低、操作快速简便等优点,适合推广应用。     

从水稻单粒糙米中快速制备基因组DNA的方法

本方法用粳稻和籼稻单粒糙米作材料制备基因组DNA,在制备过程中,加入2%CTAB破裂液直接磨碎,无需加入液氮研磨,获得浓度接近40 ng/μL,可直接用于PCR扩增的基因组DNA.与从嫩叶中提取的DNA相比,其质量无明显的差异.     

一种适用于棉花种子的DNA快速提取方法

对已报道的小麦、玉米等作物基因组DNA快速提取方法进行改良,首次在棉花上进行了尝试。以棉花种子为材料,用SDS法、NaOH法和简化法提取基因组DNA。结果表明,3种方法提取的DNA条带均较为清晰,完整性好,PCR扩增效果明显,结果稳定可靠。但DNA简化提取法整个提取过程操作简单、花费时间少、成本低,能在较短时间内完成大量样品的DNA提取。相比常规的以棉花叶片为材料的DNA提取法,本方法在降低试验成本的同时大大提高了工作效率。     

大麻基因组DNA提取及AFLP体系的建立

本研究以大麻嫩叶为材料,以改进的SDS法,提取了高质量的大麻基因组DNA,经过酶切、连接、预扩增、选择性扩增、银染等试验条件的优化,建立了AFLP反应体系。研究结果表明:在常规的SDS提取液里加入8μL/mLβ-巯基乙醇(V/V)和3%PVP(M/V)能够提取到高质量的适用于AFLP的基因组DNA;选取150ng大麻基因组DNA来进行酶切,EcoRⅠ和MseⅠ各0.5U,在37℃酶切4h,即可完全酶切。最优的选择性扩增体系为20μL反应体系中含有1.0U Taq DNA polymerase、1.0μL25mmol/L Mg2+、0.4μL10mmol/LdNTPs、50ng/μL引物各1.0μL、4.0μL稀释50倍的预扩增产物及2μL10×PCR Buffer;使用该方法,获得了清晰、稳定的图谱并筛选到了18对多态性较好的AFLP引物组合。     

珍珠黄杨DNA的提取方法比较及ISSR反应体系的优化

为从珍珠黄杨幼叶中提取高质量的总DNA,比较了1%CTAB、2%CTAB、4%CTAB和1.5%SDS 4种DNA提取方法.结果表明:2%CTAB提取的DNA A260/A280值最好,ISSR-PCR扩增效果最佳,是有效提取珍珠黄杨基因组DNA的方法.并在此基础上,以(CT)8G为引物,采用单因素实验法,确立了适合珍珠黄杨的ISSR-PCR反应体系:在总体积20 μL的反应体系中,含30 ngDNA模板,2.0 mmol/LMg2 ,0.30μmol/L引物,0.20 mmol/L dNTPs,1 U Taq酶.     

Rapid DNA Extraction method for Molecular Marker Analysis of Pennisetum Hybrid

A rapid method for DNA extraction from Pennisetum hybrid was developed.Using the method,leaves of the Pennisetum hybrid was treated by the 0.05 mol NaOH solution and heated in the boiling water for 10 min,followed by the neutralization with TE solution(ph=3.0),and the DNA extraction was used for RAPD-PCR and SSR-PCR analysis.The result indicates.The quality of the DNA by the simplified method is amount to DNA extracted by the CTAB method,and the DNA extracted by this method an meet the requirement of molecular marker analysis based on PCR technology.Combined with the 96-well plate,it can realize the high-throughput DNA extraction.     

海南龙血树基因组DNA提取方法比较

摘 要：为选择一种简单、快速、有效的海南龙血树总DNA的提取方法,分别采用CTAB法、SDS法、SDS-CTAB法和改良CTAB法提取海南龙血树嫩叶片的总DNA,用琼脂糖凝胶电泳、紫外分光光度计和ISSR对所提取的总DNA进行检测。结果表明,改良CTAB法提取的DNA A260/A280在1.7-1.9之间,纯度高、杂质少、DNA完整性好。以该方法提取的总DNA为模板进行ISSR扩增,谱带清晰,多态性、稳定性、重复性好。该法提取的总DNA适用于PCR扩增和其他分子生物学研究是有效提取海南龙血树基因组的方法。     

普洱茶发酵样品细菌和真菌DNA同时提取方法研究

本研究的目的是建立提取普洱茶发酵样品中微生物DNA的方法。首先比较了3种发酵样品中菌体收集方法,其次改进了omega小量植物DNA提取试剂盒的提取方法,以DNA的纯度、提取率和细菌16S rDNA、真菌?-微管蛋白基因PCR扩增为指标,评价提取DNA质量。实验发现茶样（5 g）加Tween-NaCl缓冲液（50 mL）,静置30 min,超声波振荡10 min,离心（10 min,12000 r/min）的菌体收集方法和在omega小量植物DNA提取试剂盒中引入液氮研磨、溶菌酶和破壁酶联合裂解细胞的方法提取的DNA纯度好,可以分别扩增出细菌16SrDNA和真菌?-微管蛋白基因。建立了普洱茶发酵样品细菌和真菌DNA同时提取方法,为开展普洱茶固态发酵过程微生物多样性的分子生物学研究奠定了基础。     

Pod photosynthesis and drought adaptation of field grown rape (Brassica napus L.)

In rape (Brassica napus L., cv. Global) seed growth mainly depends on husk CO₂ assimilation. In irrigated plants, the net photosynthetic rate (A_max) was 10–13 μmol CO₂ m⁻² s⁻¹ in non-maturing pods and correlated with nitrogen content. The stomatal conductance of water vapour (g_H2O) was 0.3 mol m⁻² s⁻¹ in non-maturing pods. The photosynthetic nitrogen use efficiency (NUE) was 8.3 μmol CO₂g⁻¹ N s⁻¹, about one-third of that in leaves. The photosynthetic water use efficiency (WUE; A_maxg_H2O⁻¹) was similar in pods and leaves. In severely droughted plants, the photosynthetic rate was reduced to 38%. The seed growth rate, however, was not influenced by intermittent periods of water stress, indicating translocation of assimilates to the seeds. The drought resistant character of the pods was due to low specific area, succulence, low stomatal conductance causing a small decrease of ΔΨ day⁻¹ during soil drying and maintenance of high relative water content during severe drought. A mathematical formulation of the pod water release curve was undertaken. © (1997) Elsevier Science B.V.     

以PCR为目的的大豆叶片DNA提取方法的比较研究

模板DNA的质量直接影响PCR扩增的结果,而不同提取方法及其缓冲液的成份与浓度对提取DNA的质量有重要影响。本文以5个栽培大豆品种的叶片为材料,比较分析了SDS与CTAB两种提取方法以及不同浓度CTAB提取缓冲液对所提取的DNA质量的影响,并通过PCR进行检验。实验结果表明：用1％(W/V)、2％(W/V)浓度的CTAB提取缓冲液和1．25％(WV)SDS提取缓冲液所提取的大豆叶片DNA的质量较好,均能满足PCR扩增模板的需求,其中以1．25％(W／V)SDS提取得到的大豆叶片DNA质量最好,以其为模板扩增的效果最佳,而4％浓度的CTAB不适宜提取大豆叶片DNA。     

Evaluation of 'quick and dirty' DNA extraction methods for marker-assisted selection in rice (Oryza sativa L.)

Six simple methods for extracting DNA from rice seedlings were evaluated for marker‐assisted selection (MAS). The assessment of each method was based on PCR amplification of SSR markers, DNA yield and purity, time and cost. Based on these criteria, two methods were selected as being superior to other methods. The best two methods included the standard method developed at the International Rice Research Institute (IRRI), which utilizes a sodium dodecyl sulfate extraction buffer followed by chloroform/isoamyl alcohol extraction and a previously published method using sodium hydroxide and Tris. These two methods produced nearly identical PCR amplification results. The sodium hydroxide method is considerably simpler, quicker and cheaper than the standard IRRI method, and may be particularly useful for many applications of MAS or high‐resolution mapping. This method was also adapted into an effective high throughput method utilizing 96‐well plates emphasizing its versatility.     

一种适用于PCR扩增的甜菜干种子DNA快速提取方法的研究

为了寻求一种快速提取甜菜DNA的方法,利用改良CTAB法对甜菜干种子DNA进行了提取,经琼脂糖电泳及SRAP和SSR两种随机引物扩增检测,结果表明该法提取的DNA完整性好,SSR及SRAP扩增条带清晰,符合甜菜SSR-PCR及SRAP-PCR对DNA质量的要求。本研究建立了快速提取甜菜DNA的体系,为分子标记在将来甜菜品种指纹图谱构建及辅助育种等方面的应用奠定了坚实的基础。     

利用PCR仪快速提取甜菜基因组DNA

为了寻找一种快速提取甜菜基因组DNA的方法,以甜菜干种子、幼苗、种仁以及甜菜叶片干粉为原料,利用PCR仪结合碱裂解法快速提取甜菜基因组DNA,利用微量分光光度计检测取DNA的浓度,并用甜菜SSR引物对提取的DNA进行扩增。结果表明,在4 种材料中均检测到了DNA,干种子、幼苗、种仁以及叶片干粉中提取的DNA平均浓度分别为432、197、158、448 ng/μL,无论是DNA原液还是稀释到20 ng/μL的工作液,均能在SSR-PCR反应中扩增出清晰的条带。该方法提取甜菜基因组DNA简单、快速,仅需要NaOH和HCl两种药品,提取的DNA完全可以用于SSR-PCR反应,为快速鉴定甜菜品种纯度和真实性提供了技术支持。     

Extraction of Cotton Fiber DNA and Its Application in Traceability

[Objective] To address the questions of lacking monitoring system for origins of varieties, this study explored a DNA extraction method suitable for cotton fiber, aiming to establish an identification system that uses cotton fiber DNA as a medium to trace the authenticity and cultivar of cotton varieties. [Method] By improving and optimizing the extraction method, the DNAs of cotton fibers at different days post anthesis and lint from different years were extracted and used as templates for routine polymerase chain reaction (PCR) amplification. 13 pairs of simple sequence repeat(SSR) primers were selected for SSR-PCR amplification using the DNA of Lu 1127, Shikang 126 and Ruiza 816. [Result] The DNA extraction method can extract cotton fiber DNAs of different developmental stages and different years. With the development of fiber, although the extracted DNA content is reduced, it can still meet the needs of downstream experiments. The extracted cotton fiber DNAs can be subjected to conventional PCR amplification, the PCR amplified bands were clear; 13 pairs of cotton SSR primers were used for SSR-PCR amplification of cotton fiber DNAs of Lu 1127, Shikang 126 and Ruiza 816, and a clear polymorphic band could be amplified and easily distinguished. [Conclusion] This extraction method is suitable for extracting genomic DNA of cotton fiber, and is satisfied with conventional PCR amplification with SSR markers. It is confirmed that the cotton fiber DNA molecular markers are feasible for tracing the source of cotton varieties, and it is expected to provide technical support for ensuring the safety of cotton industry.