共查询到19条相似文献，搜索用时 203 毫秒
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用正交设计研究了DNA聚丙烯酰胺凝胶电泳(PAGE)凝胶的简易银染中各因素对银染效果的影响,通过PCR技术扩增了山西白猪第六世代15个耳组织基因组的两条微卫星DNA序列,这些序列经聚丙烯酰胺凝胶电泳分离和500ladder Marker标记跑板后,利用正交法设计简易银染法各因素的组合,对凝胶进行染色。试验结果表明,染色液用AgNO3浓度0.10%、显色用含NaOH1.50%、甲醛0.40%混合液、水温30℃时对PAGE凝胶染色的效果最好。 相似文献
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《中国畜牧兽医文摘》2012,(10)
<正>用2个微卫星标记,通过PCR扩增,利用12%非变性聚丙烯酰胺凝胶电泳和银染显色等方法对河南淮山羊进行微卫星DNA遗传多态性进行研究。结果显示:在Bulge5基因座上检测出6个等位基因,其片段大小在105～140bp之间,其多态信息含量(PIC)、有效等位基因数(Ne) 相似文献
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利用公布的家蚕基因组序列，设计特定的家蚕微卫星引物，对8个不同品种家蚕的微卫星位点进行PCR扩增，所得PCR产物经变性与非变性聚丙烯凝胶（PAGE）分离，银染后发现在非变性PAGE上，条带较粗且模糊，非特异带较多，导致读带误差增大；而在变陛PAGE上，所得条带较细且比较清晰，非特异带明显减少，有利于降低统计误差。 相似文献
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从鸡血中快速提取高质量基因组DNA方法的研究 总被引：19，自引：1，他引：19
介绍了一种经改进建立的从鸡血中快速提取高纯度基因组DNA的方法。该方法提取DNA耗时较短,只需2.5~3 h即可拿到DNA样品。提取的DNA样品经紫外分光光度计检测,DNA样品的A260/A280达1.782 2±0.009 3;经琼脂糖凝胶电泳检测,DNA样品的带型清晰、整齐,无拖尾现象;微卫星PCR扩增效果理想。结果表明,该方法所提DNA纯度较高,质量好,分子片段完整,完全能满足分子生物学实验的要求。 相似文献
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微卫星电泳及银染检测中的常见问题分析及对策 总被引：1，自引：0，他引：1
微卫星DNA,又称短串联重复序列(short tandemrepeat STR),是一类广泛存在于原核/真核生物基因组的DNA串联重复序列。它是继RFLP之后兴起的一种新的分子遗标记技术。因其多态信息含量高,检测快速,重组率低,已被越来越多的应用于基因定位及构建基因组图谱和群体遗传结构分析及杂交优势预测等诸多方面[1]。尽管微卫星标记方法操作简便、快捷,但因其灵敏度高、操作时间短,在实际操作中出现的一些问题常常会影响最终的检测结果。笔者总结了一年来STR工作中所遇到的一些有关于微卫星电泳及银染的常见问题,做出了相应的分析和对策,对于保证… 相似文献
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以产志贺毒素样大肠杆菌（SLTEC）F18ab血清型标准菌株107/86基因组DNA为模板,利用PCR技术成功扩增出编码F18ab完整菌毛操纵子fed基因,克隆入表达载体pBR322,经限制性内切酶酶切分析,DNA琼脂糖电泳鉴定并结合序列测定分析,构建和筛选出含fed完整基因正确插入的pBR322-fed重组质粒,将上述重组质粒转化至不含任何菌毛结构的大肠杆菌SE5000,该表达重组菌能分别与兔抗F18ab亚单位蛋白FedF高免血清、鼠抗F18ab菌毛a单因子单克隆抗体、兔抗F18ab菌毛高免血清和抗F18ab菌毛IgG抗体产生明显的凝集反应。用热抽提法分别抽提和纯化SLTEC F18ab标准株107/86和重组菌SE5000（pBR322-fed）体外表达的F18ab菌毛,纯化菌毛经SDS-PAGE电泳和考马斯亮蓝染色获单一相对分子质量约为15 000蛋白条带。Western-blotting结果表明：兔抗F18ab菌毛高免血清能特异性识别SLTEC F18ab标准株107/86和重组菌SE5000（pBR322-fed）所提纯的单一主要结构蛋白。用重组菌SE5000（pBR322-fed）进行易感仔猪小肠上皮细胞体外黏附试验和黏附抑制试验,结果表明：重组菌SE5000（pBR322-fed）和SLTEC F18ab标准株107/86一样具有较强的黏附易感仔猪小肠上皮细胞的能力,而兔抗F18ab菌毛高免血清能有效地抑制上述重组菌SE5000（pBR322-fed）和SLTEC F18ab标准株107/86对易感仔猪小肠上皮细胞的黏附结合。 相似文献
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PRV闽A株Bam HI片段克隆及其第7片段的鉴定 总被引：4，自引：2，他引：2
用鸟枪法将ＰＲＶ闽Ａ株的ＢａｍＨＩ酶切片断克隆到ｐＢＲ３２２质粒中，再经抗性筛选、酶切鉴定和菌落原位杂交，证实已克隆了ＰＲＶ闽Ａ株１４个ＢａｍＨＩ酶切片段中的１２个，从而构建了其基因文库，通过Ｓｏｕｔｈｅｒｎ转印杂交和酶切图谱分析鉴定了重组质粒ｐＰＲ１２８，其插入片段含量包含了ＰＲＶ闽Ａ株糖蛋白ｇｐ５０基因在内的ＢａｍＨＩ－７片段，核酸长约６．８ｋｂ。 相似文献
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An Anaplasma marginale DNA probe has been developed by using an improved method for the isolation of genomic DNA. Purified genomic A. marginale DNA from the St. Croix isolate was partially digested with Sau 3A1 into fragments (greater than or equal to 5.0 kb). The restriction fragments were cloned using standard techniques in the pBR322 vector and used to transform E. coli (DH5) host cells. The recombinant A. marginale DNA library was screened by the colony lifting procedure. Colonies containing plasmids with A. marginale DNA inserts were identified by hybridization with a genomic A. marginale DNA radiolabeled probe (32P). Seven recombinant A. marginale DNA probes were evaluated by dot-blot in vitro hybridization assays to identify candidates as diagnostic tools in bovine anaplasmosis studies. Specificity and sensitivity experiments were carried out by using heterologous and homologous DNAs. The heterologous panel contained bovine DNA (WBC) and blood parasites DNA from Babesia bovis (Bb), Babesia bigemina (Bbi), Eperythrozoon suis (Es) and Eperythrozoon wenyoni (Ew). The homologous DNA panel included A. marginale DNAs of 12 different isolates which were isolated in the Caribbean, Mexico, and the U.S.A. The selected diagnostic probe was identified as pSt. Croix A1, and labeled with 32P by using in vitro nick translation and random primer techniques. The pSt. Croix A1 probe demonstrated 100% specificity and high sensitivity by hybridization in dot blotting and Southern blotting. The probe can detect 500-1000 infected erythrocytes per microliters which corresponds to a parasitemia of less than 0.01%. The A. marginale DNA insert was approximately 6.4 kb in size and a partial restriction map has been constructed. 相似文献
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K.R.E. Squire R.Y. Chuang B.I. Osburn D.L. Knudson R.H. Doi 《Veterinary microbiology》1983,8(6):543-553
Various double-stranded RNA extraction procedures, gel electrophoresis systems, and methods to detect the RNA bands in the gel were investigated to find the most rapid methods to obtain the genome profiles of bluetongue virus in small volumes (1–25 ml) of infected cell culture fluids. Rapid double-stranded RNA extraction procedures coupled with staining the acrylamide gel slabs with ethidium bromide or silver nitrate resulted in well-defined genome profiles from bluetongue virus infected cell cultures in 6–48 h. Radioactive labelling of viral RNA with 32P was time consuming, cumbersome and expensive. These techniques detect less than 0.5 μg of double-stranded RNA which can be obtained from one 1-ml well of a 24-well cluster plate of bluetongue virus infected cell monolayers. The methods were therefore suitable for rapid comparisons of the electropherotypes of multiple virus isolates. 相似文献
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《Journal of aquatic animal health》2013,25(4):246-253
Abstract Lipopolysaccharide (LPS) was purified from 40 isolates of Edwardsiella ictaluri by two methods: (1) enzyme digestion and hot aqueous phenol extraction and (2) enzyme digestion and gel exclusion chromatography. Purified LPS was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and by immunoblot analysis. Both methods of purification yielded smooth LPS as evidenced by a ladderlike pattern of more than 40 LPS bands. With silver staining, both low- and high-molecular-mass LPS bands were seen. Lower-molecularmass LPS stained more intensely than higher-molecular-mass LPS bands, indicating a preponderance of lower-molecular-mass LPSs. Lipopolysaccharide bands from the 40 isolates migrated similarly within SDS-PAGE gels, indicating a high degree of structural similarity among the isolates examined. The ladderlike array was more easily seen with immunoblot analysis than with silver staining of SDS-PAGE gels. Additionally, immunoblot analysis revealed a high degree of antigenic similarity among the 40 isolates. 相似文献