共查询到20条相似文献，搜索用时 170 毫秒
1.
采用改进的CTAB法提取中国特有濒危裸子植物白豆杉(Pseudotaxus chienii)嫩叶总DNA,进行RAPD分析.分别研究了模板DNA、引物、dNTPs、Mg2 和TaqDNA聚合酶用量对RAPD PCR扩增反应结果的影响.筛选并建立了适合于白豆杉RAPD扩增的反应体系:总体积25μL,其中10×PCR缓冲液(500 mmol/L KCl,100 mmol/L Tris-HCl,1.0%Triton X-100,20 mmol/L MgCl2)2.0μL,引物(1.5μmol/L)0.5μL,dNTPs(10 mmol/L)0.4μL,模板1μL(35 ng),Taq酶0.8μL(0.5 U),水20.3 μL. 相似文献
2.
采用改进的CTAB法提取中国特有濒危裸子植物白豆杉(Pseudotaxus chienii)嫩叶总DNA,进行RAPD分析.分别研究了模板DNA、引物、dNTPs、Mg2 和TaqDNA聚合酶用量对RAPDPCR扩增反应结果的影响.筛选并建立了适合于白豆杉RAPD扩增的反应体系:总体积25μL,其中10×PCR缓冲液(500mmol/L KCl,100mmol/L Tris-HCl,1.0%Triton X-100,20mmol/L MgCl2)2.0μL,引物(1.5μmol/L)0.5μL,dNTPs(10mmol/L)0.4μL,模板1μL(35ng),Taq酶0.8μL(0.5U),水20.3μL. 相似文献
3.
用SDS法提取了苜蓿基因组DNA的模板。从100个10bp的随机引物中筛选出了12条能在11个苜蓿品种中均能扩增出清晰的片段的引物。应用L16(45)正交表,研究了DNA、dNTPs、Mg2 、Taq酶和引物的浓度对扩增迁移率重现性的影响。用这种方法建立的苜蓿RAPD-PCR优化反应体系为:20μL体系中含DNA模板的浓度是5.0mg/L、dNTPs浓度是0.175mmol/L、Mg2 浓度是1.25mmol/L、Taq酶在20μL的体系中是1.00U、引物浓度是0.40μmol/L。 相似文献
4.
枇杷属植物RAPD反应体系的优化与应用 总被引：2，自引：0，他引：2
采用CTAB—蛋白酶K法提取了48份枇杷种质的DNA,以西班牙枇杷品种Ullera为模板DNA,对枇杷属植物RAPD反应体系进行优化研究,结果表明:25μL反应体系中,Taq DNA聚合酶、Mg2+、引物、模板DNA和dNTPs等5种主要成分的适宜浓度或用量分别是:Mg2+为2.0 mmol/L,Taq DNA聚合酶为1.0 U,引物为0.25"mol/L,dNTPs为0.100—0.125 mmol/L,模板DNA为25—30 ng。利用该优化体系对48份枇杷种质进行RAPD随机扩增,经琼脂糖电泳获得了清晰的扩增图谱。 相似文献
5.
红松SRAP-PCR反应体系的建立及优化 总被引：1，自引：0，他引：1
相关序列扩增多态性(Sequence-Related Amplified Polymorphism,SRAP)是对ORFs(OpenReading Frames)进行扩增的一种新型的基于PCR的标记方法。以CTAB法提取的红松针叶DNA为模板,应用单因子试验及L16(45)正交试验系统分析了DNA模板浓度、Mg2+浓度、dNTPs浓度、引物浓度、Taq酶浓度等对SRAP-PCR扩增结果的影响。确定了PCR的最佳反应体系为:20μL体系中,1×PCRbuffer 2μL、DNA模板40~100 ng,Mg2+2.5mmol/L,dNTPs 0.15 mmol/L,引物0.15μmol/L,Taq酶1.5 U。PCR最优的扩增程序为:进行35个循环,前5个循环中94℃变性1 min,35℃复性1 min,延伸30 s;后30个循环中94℃变性1 min,50℃复性1 min,72℃延伸1 min;最后72℃延伸7 min,4℃保存。在此反应体系下,所扩增谱带清晰、稳定、多态性高,为进行红松不同种源间遗传分化以及构建遗传图谱等内容的研究奠定基础。 相似文献
6.
采用单因子梯度试验法对影响雷公藤RAPD—PCIK反应的Mg^2＋浓度、dNTPs浓度、引物浓度、Taq DNA聚合酶的用量及DNA模板浓度进行了筛选。结果表明获得清晰、重复性高的雷公藤RAPD-PCR扩增条件为：20μL PCR反应体积中,2．0mmol·L^-1 MgCl2,0．2mmol·L^-1 dNTPs,0．2μmol·L^-1引物,50ng模板DNA,1UTaq DNA聚合酶。 相似文献
7.
本研究以辣椒基因组DNA为模板,优化辣椒SRAP反应体系中的参数,建立了一套适用于辣椒的SRAP-PCR最佳反应体系。在25μL反应体系中,模板DNA 50 ng、上下游引物各0.1μmol/L、dNTPs 0.1mmol/L、Taq DNA聚合酶1.5 U、Mg2+浓度为2.0 mmol/L。扩增程序为:94℃3 min;94℃30 s、35℃30 s、72℃1.5 min,5 cycles;94℃30 s、54℃30 s、72℃30 s,35 cycles;72℃10 min,用2%琼脂糖凝胶电泳和8%非变性聚丙烯酰胺凝胶电泳检测扩增结果。利用该优化体系筛选引物,筛选出条带清晰、重复性好的15对SRAP引物组合,验证了体系的实用性。 相似文献
8.
正交设计在梨基因组RAPD优化体系中的应用 总被引：2，自引：0，他引：2
采用正交设计研究方法,建立了梨RAPD分析的PCR反应体系,成功地进行了RAPD扩增,筛选出梨RAPD的最佳方案为:25μl反应体系中包括引物0.5μmol.μL-1,dNTPs0.1mmol.L-1,模板DNA10ng,Taq酶1U,Mg2 4mmol.L-1,10buffer缓冲液2.5lμ,其余部分用无菌超纯水补平。PCR扩增循环为95℃3min,38℃1min,72℃2min1次循环;94℃1min,38℃1min,72℃2min,46次循环,最后72℃延伸10min。 相似文献
9.
以改进SDS法抽提濒危植物水杉嫩叶总DNA,进行随机扩增多态DNA(RAPD)分析,分别测试了镁离子,dNTP,模板DNA含量,引物和DNA聚合酶量对反应结果的影响,通过各因子的组合研究,可知水杉RAPD分析较适宜的扩增条件是:15μlPCR反应体积,1×Taq酶配套缓冲液(10mmol/LTris HCl pH 9,0.50mmol/L KCl,0.1％Triton X-100),1.5mmol/L MgCl_2,0.5U Taq酶(上海华美公司),5ng模板DNA,20pmol引物(上海Sangon公司);2μg/μlBSA;dATP、dCTP、dGTP、dTTP各0.2mmol/L。 相似文献
10.
11.
建立并优化虎耳草ISSR-PCR反应体系和扩增程序,为探讨虎耳草种质资源遗传多样性奠定基础.采用正交设计方法和单因子试验,研究TaqDNA聚合酶、dNTP、Mg2+、引物、模板DNA、延伸时间及循环次数对PCR扩增的影响.结果表明:虎耳草ISSR-PCR的最佳反应体系为:在25μL的反应体系中含模板DNA 35ng,Mg2+1.25mmo/lL、dNTP 380μmo/lL、引物1.2μmo/lL、Taq DNA聚合酶1.5U.反应程序为:95℃预变性5min;94℃变性1min,50℃(据不同引物的退火温度)退火70s,72℃延伸1.5min,40个循环;72℃延伸10min,4℃保存.经过11份虎耳草种质检验,证明该体系稳定可靠,可用于虎耳草种质资源遗传多样性分析及居群鉴别的研究. 相似文献
12.
本研究以NS5-NS6为引物,扩增真菌同源性序列ssu-rDNA片段,建立广范围真菌检测的方法。用此方法扩增医学主要致病真菌、细菌和人体细胞,结果所有真菌均扩增出一个约310bp的产物,而细菌和人体细胞均扩增阴性,1pg白色假丝酵母菌DNA即可检出。 相似文献
13.
利用聚合酶链式反应(PCR)方法特异性扩增河北省饶阳县大仓鼠(Circetulvs triton)基因组DNA通过琼脂糖凝胶电泳检测以期出现特异性条带，可以进一步回收进行序列分析． 相似文献
14.
ＩＮＴＲＯＤＵＣＴＩＯＮＦｌｕｏｒｅｓｃｅｎｃｅｉｎ ｓｉｔｕｈｙｂｒｉｄｉｚａｔｉｏｎ (ＦＩＳＨ )ａｎｄｐｏｌｙｍｅｒａｓｅｃｈａｉｎｒｅａｃｔｉｏｎ (ＰＣＲ)ｈａｄｂｅｅｎｓｕｃｃｅｓｓｆｕｌｌｙｕｓｅｄｆｏｒｐｒｅｉｍｐｌａｎｔａｔｉｏｎｇｅｎｅｎｔｉｃｄｉ ａｇｎｏｓｉｓ (ＰＧＤ ) (Ｖｅｒｌｉｎｓｋｙｅｔａｌ.,1 997,Ｓａｂｔａｌｏｅｔａｌ.1 995)ａｌｔｈｏｕｇｈｔｈｅｙｎｏｒｍａｌｌｙｏｎｌｙｏｆｆｅｒｉｎｆｏｒｍａｔｉｏｎｆｒｏｍｏｎｅｏｒｆｅｗｃｈｒｏｍｏｓｏｍｅｒｅ ｇｉｏｎｓｏｒｓｐ… 相似文献
15.
以渐危植物海菜花基因组DNA为研究对象,筛选引物UBC-818【序列为（CA）8G】研究并确定了该引物适宜的退火温度,对影响ISSR扩增效果的主要参数进行筛选和优化,确定了海菜花的ISSR分析的最佳反应体系：15μL反应体系中含25ng模板DNA,0．2mmol·L^-1引物,0．2μmol·L^-1dNTPs,2．0mmol·L^-1Mg^2＋,0．5UTaq酶,1．5μL10×PCRBufferMg2＋（-）[100mmol·L^-1Tris—HCl（pH8．3）,500mmol·L^-1KCl]． 相似文献
16.
Wang JY Gu YS Wang J Tong Y Wang Y Shao JB Qi M 《Journal of Zhejiang University. Science. B》2008,9(8):610-615
Objective:Leber's hereditary optic neuropathY (LHON)is a maternally inherited degeneration of the optic nerve caused by point mutations of mitochondrial DNA(mtDNA).Many unsolved questions regarding the penetrance and pathophysiological mechanism of LHON demand efficient and reliable mutation testing.This study aims to develop a minor groove binder(MGB) probe assay for rapid detection of mtDNA11778 mutation and heteroplasmy in Chinese LHON patients by real-time polymerase chain reaction(PCR).Methods:Forty-eight patients suspected of having LHON and their maternal relatives underwent a molecular genetic evaluation,with 20 normal individuals as a control group at the same time.A real-time PCR involving two MGB probes was used to detect the mtDNA 11778 mutation and heteroplasmy.A linear standard curve was obtained by pUCmLHONG and pUCmLHONA clones.Results:All 48 LHON patients and their matemal relatives were positive for mtDNA 11778 mutation in our assay,27 heteroplasmic and 21 homoplasmic.Eighteen cases did not show an occurrence of the disease,while 9 developed the disease among the 27 heteroplasmic mutation cases.Eleven did not show an occurrence of the disease,while 10 cases developed the disease among 21 homoplasmic mutation cases.There was a significant difierence in the incidence between the heteroplasmic and the homoplasmic mutation types.The time needed for running a real-time PCR assay was only 80 min.Conclusion:This real-time PCR assay is a rapid,reliable method for mtDNA mutation detection as well as heteroplasmy quantification.Detecting this ratio is very important for predicting phenotypic expression of unaffected carriers. 相似文献
17.
都支杜鹃ISSR扩增条件的优化 总被引：1，自引：0，他引：1
对珍稀濒危物种都支杜鹃的ISSR扩增体系中的Mg2+浓度、dNTP浓度、Taq酶含量和底物含量进行优化,并在优化后的基础上筛选出8条效果较好的引物,在梯度PCR仪中设置温度梯度,找到这些引物的最佳退火温度。优化结果表明,至少在一定的范围内底物浓度和Taq酶含量对PCR反应结果影响不大,相对而言,dNTP浓度和Mg2+浓度对反应结果的影响更加显著。优化后的15 ul PCR反应体系中包括20 ng模板DNA、0.3μM引物、1.5 ul Buffer(Mg2+free),0.5 U Taq酶、1.5 mM MgCl2和0.1 mM dNTP。 相似文献
18.
针禾属植物羽毛针禾基因组DNA提取方法的研究 总被引：3，自引：0，他引：3
传统的CTAB DNA提取法步骤多.较烦琐,DNA产率低,而且由于酚、单宁、色素、多糖很难完全去除,容易影响随机扩增多态、微卫星等标记工作的效率.采用SDS裂解法提取了针禾属植物幼嫩叶片的基因组DNA,所获得的DNA可用于PCR反应.并且在针禾属植物的研究中得到了较好的结果. 相似文献
19.
Qi WANG Xiaoxia SHEN Tian QIU Wei WU Lin LI Zhi'an WANG Huixia SHOU 《Journal of Zhejiang University. Science. B》2021,22(2):99
Nucleic acids in plant tissue lysates can be captured quickly by a cellulose filter paper and prepared for amplification after a quick purification.In this study,a published filter paper strip method was modified by sticking the filter paper on a polyvinyl chloride resin(PVC)sheet.This modified method is named EZ-D,for EASY DNA extraction.Compared with the original cetyl trimethylammonium bromide(CTAB)method,DNA extracted by EZ-D is more efficient in polymerase chain reaction(PCR)amplification due to the more stable performance of the EZ-D stick.The EZ-D method is also faster,easier,and cheaper.PCR analyses showed that DNA extracted from several types of plant tissues by EZ-D was appropriate for specific identification of biological samples.A regular PCR reaction can detect the EZ-D-extracted DNA template at concentration as low as 0.1 ng/μL.Evaluation of the EZ-D showed that DNA extracts could be successfully amplified by PCR reaction for DNA fragments up to 3000 bp in length and up to 80%in GC content.EZ-D was successfully used for DNA extraction from a variety of plant species and plant tissues.Moreover,when EZ-D was combined with the loop-mediated isothermal amplification(LAMP)method,DNA identification of biological samples could be achieved without the need for specialized equipment.As an optimized DNA purification method,EZ-D shows great advantages in application and can be used widely in laboratories where equipment is limited and rapid results are required. 相似文献
20.
Detection of PCV2 DNA by SYBR Green I-based quantitative PCR 总被引：5，自引：0，他引：5
We developed an assay for the detection and quantitation of porcine circovirus type 2 (PCV2) with the SYBR Green I-based real-time PCR. The real-time PCR provides a broad dynamic range, detecting from 103 to 1011 copies of DNA per reaction. No cross-reactions were found in specimens containing PCV1. Because of the high sensitivity and specificity of the assay with a relatively rapid and simple procedure, real-time PCR can be used as a routine assay for the clinical diagnosis of PCV2 infection. In this study we applied real-time PCR assay to 80 clinical samples, collected from 40 pigs with postweaning multisystemic wasting syndrome (PMWS) and 40 healthy pigs in comparison with conventional PCR assay. In 56 of 80 samples, PCV2 DNA was de-tected by conventional PCR assay. All samples positive for PCV2 DNA in conventional PCR assay were also positive in real-time assay, and 12 of 24 samples that tested negative for PCV2 DNA in the conventional assay were tested positive in real-time PCR assay. Real-time PCR assay increased the number of samples in which PCV2 was detected by 15%. It is, therefore, considered to be a useful tool for the detection of PCV2. 相似文献