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1.
介绍两类从普通琼脂糖电泳凝胶中回收DNA的简便,快捷,高效且廉价的方法,第一类为电泳洗脱法,方法a.利用1.5mL微量离心管,1mL吸头,尼龙网膜和透析膜做成的一个小装置,快速有效回DNA,最终回收率为70%左右,方法b:不用DEAE-纤维素膜,而用透析膜在凝胶中作出横隔挡在DNA条带前,最终回收率为50%左右,第二类为冰冻融解法,最终回收率也在50%左右,如果联合使用冰冻融解法和电泳洗脱法,回收  相似文献   

2.
我们设计了一种简单电洗脱装置,从琼脂糖胶中回收DNA。该装置由两个带旋盖的小管、两块透析膜和一个凝胶屏障组成。在电场作用下,DNA从凝胶中迁移出来,通过凝胶屏障进入由凝胶屏障和透析膜组成的回收小仓。用微量吸样器收集DNA,乙醇沉淀和清洗。该法DNA的回收率约85%;回收的DNA可用于基因工程常规实验。  相似文献   

3.
已知分子量大小的染色体DNA分子标记是脉冲电泳研究中用以估算样本染色体DNA分子大小必不可少的参照物。对用做标记的啤酒酵母(Sacharomycescerevisiae)和粟酒裂殖酵母(Schizosacharomycespombe)完整染色体DNA几种制备方法的比较研究以及改进研究表明,液氮冷冻研磨法,凝胶包埋法以及凝胶包埋破壁法效果均很好,都得到大量染色体DNA,且彼此间无明显差异。表明液氮冷冻研磨法和凝胶包埋法制备上述两种酵母菌染色体DNA分子标记是两种理想的方法,可完全取代凝胶包埋破壁法,即缩短处理时间又降低成本。此外,相同的电泳样本块在不同的电泳条件下,所得结果有一定的差异,表明电泳条件是影响电泳结果的一个重要因素  相似文献   

4.
SOD的聚丙烯酰胺梯度凝胶电泳分离和酶活性显示   总被引:3,自引:0,他引:3  
SOD的聚丙烯酸胺凝胶电泳技术已被广泛应用于动、植物SOD同工酶的研究。我们改用浓度梯度胶电泳法分离和鉴定SOD,其谱带受杂蛋白干扰少,分辨率和灵敏度比均一胶电泳法高,并能根据电泳相对迁移率较为快速和准确地初步判定SOD同工酶类型。现简介如下:门)SOD浓度梯度胶的制作基本按张龙翔等方法(生化实验方法和技术,北京:人民教育出版社,1982.119),凝胶梯度为4%~35%。制成的凝胶先预电泳除去残留的过硫酸按,点样后在4C左右电泳,一般SOD港带达到相对稳定需3500~4000V/h。(2)SOD活性染色按罗广华、王爱国方法[植…  相似文献   

5.
利用RT-PCR方法成功地从PI(PMA,Ionomycin)、PHA活化的人T细胞中扩增出hIL-17编码区cDNA(468bp),克隆测序证实得到该基因.用特殊设计的引物扩增hIL-17成熟肽编码区(414bp),接入表达载体pET30a质粒中.pET30a-mhIL-17在大肠杆菌BL21(DE3)中获得高效表达,目的蛋白占总菌体蛋白50%左右.经凝胶过滤和阴离子交换层析两步分离纯化和复性,得到单体型rmhIL-17,纯度达98%以上,N端16个氨基酸序列分析,结果与文献报道一致.SDS-PAGE法测定分子量为16kD,薄层丙烯酰胺凝胶等电聚焦分析该蛋白等电点为pH8.8~8.9,3HTdR参入法测定rmhIL-17单体的体外活性,实验结果表明rmhIL-17对PHA活化人的PBMC细胞具有抑制作用,结果同可溶型IL-17R性质相似  相似文献   

6.
本文根据文献和笔者近年来的研究工作,分述电泳后凝胶中核酸的洗脱、回收方法及其优缺点,以供从事这方面工作的同行参考。一、电泳回收法(一)柱状电泳洗脱法1.从聚丙烯酰胺凝胶中洗脱:Allet等于1973年报告以内切酶RI切割λDNA,用梯度  相似文献   

7.
冯博  李育阳 《遗传》1989,11(3):41-42
分离与回收DNA片段是基因操作的重要环节之一。本文介绍了一个用透析膜从琼脂糖胶中回收 DNA 片段的改进方法。利用本法回收DNA片段洗脱容易、节约时间、回收率在80% 左右。回收的 DNA片段可用于酶切反应、连接反应和用缺口位移反应制备32p标记DNA探针。  相似文献   

8.
目的:通过测定rhIGF-1等电点为例,建立一种快速测定蛋白质等电点的方法。方法:采用等电聚焦电泳(isoelectric focusing,IEF)和高效液相色谱(HPLC)连用的方法,主要以7.5%的凝胶浓度、3%的交联度及6%的两性电解质浓度配置凝胶,电泳后的凝胶经凝胶成像系统和HPLE分析确定目的条带及其pI值。结果:rhIGF-1的等电点为pI=8.20,pH梯度曲线线形回归方程为Y=0.0664X+3.8217,相关系数r达0.9956,说明等电聚焦电泳测定结果准确。  相似文献   

9.
用做标记的两种酵母菌完整染色体DNA的简化制备法   总被引:3,自引:2,他引:1  
蒋继志  尚晓冬 《菌物系统》1998,17(2):174-178
已知分子量在小的染色体DNA分子标记是脉冲电泳研究中用以估算样本染色DNA分子大小必不可和的参照物。对用做标记的啤酒酵母和粟酒裂殖酵母完整染色体DNA几种制备方法的比较研究以及改进研究表明,液氮冷冻研磨法,凝胶包埋法以及凝胶包埋破壁法效果均很好,都得到大量染色体DNA,且彼此此间无明显差异。  相似文献   

10.
PCR-SSCP分析方法的优化   总被引:3,自引:0,他引:3  
王娜  连易水  刘砚星 《现代生物医学进展》2008,8(10):1841-1844,1825
目的:优化PCR-SSCP分析方法。方法:选择野生型Kir2.3质粒和突变型Kir2.3(2123L)质粒为样本,其PCR结果的鉴定采用2%的琼脂糖凝胶EB显色,SSCP分析采用12%的非变性聚丙烯酰胺凝胶DNA银染显色。PCR产物变性比较了三种方法:即碱变性、SDS变性、直接变性。聚丙烯酰胺凝胶根据丙烯酰胺和甲叉双丙烯酰胺及甘油的比例设计了六个实验组,每组采用两种条件电泳,即:30mA恒流快速电泳和100V恒流过夜电泳。结果:选择直接变性的PCR产物作为变性上样液,并确定了三种凝胶配及该条件下适用的电泳条件。即:丙烯酰胺与甲叉双丙烯酰胺的比例为49比1,凝胶中含1%甘油,4℃,500V高压电泳3min接着30mA恒流快速电泳3h;丙烯酰胺与甲叉双丙烯酰胺的比例为49比1,凝胶中含5%甘油,4℃,500V高压电泳3min接着30mA恒流快速电泳3h;丙烯酰胺与甲叉双丙烯酰胺的比例为29比1,凝胶中不合甘油,4℃,500V高压电泳3min接着100V恒流过夜电泳12h。结论:条件优化的PCR-SSCP是一种筛查基因突变简便、有效的实验方法。  相似文献   

11.
简便实用的琼脂糖凝胶回收DNA片段方法   总被引:8,自引:0,他引:8  
介绍一种简便实用的DNA片段回收方法,与以前所报道的DEAE-纤维素膜电泳法、透析袋电洗脱法、低融点琼脂糖凝胶法、凝胶冻融法等相比,所需器材简单、操作简便、回收率高、成本低。回收的DNA片段在进一步克隆和测序中表现出较好的效果,是一种适合于科研和教学的实验方法。  相似文献   

12.
该文研究了蜘蛛大分子量基因组DNA(HMW-gDNA)的提取以及一种高效电洗脱纯化装置的构建。以蜘蛛胸部肌肉组织为原料,通过自改良CTAB法提取蜘蛛HMW-gDNA,利用透析膜和2 mL离心管构建一种新的HMW-gDNA快速凝胶回收装置,并对蜘蛛HMW-gDNA进行电洗脱分离回收。结果显示,改良CTAB法可高效提取蜘蛛HMW-gDNA(>48.5 kb),且通过透析膜的截留作用,对普通琼脂糖凝胶中目的HMW-gDNA进行快速电洗脱分离,其回收率超过75%,OD260/OD280处于1.8~2.0之间,对HMW-gDNA完整性无影响。综合结果表明, 改良CTAB法可用于蜘蛛HMW-gDNA的提取,此电洗脱纯化装置可从普通琼脂糖中高效回收HMW-gDNA,是一种低成本、简捷、高效且实用性强的凝胶回收方法。  相似文献   

13.
A rapid and inexpensive method for the electroelution of DNA fragments from agarose gels is described. DNA fragments were separated by agarose gel electrophoresis and visualized by staining with ethidium bromide. Selected DNA fragments were placed into electroeluter tubes capped with dialysis membrane and electroeluted into a small volume of buffer using a conventional horizontal gel electrophoresis apparatus. The method successfully eluted and concentrated DNA fragments with molecular weights ranging from 2.7 to 13.9 MDa in 3 h.  相似文献   

14.
Electroelution of DNA and protein from polyacrylamide and agarose gels   总被引:1,自引:0,他引:1  
An electroelution method is described for the recovery of DNA and protein from agarose or polyacrylamide gels. The samples to be electroeluted are compartmentalized in a modified microcentrifuge tube fitted with dialysis membranes. This procedure is simple, rapid, inexpensive and efficient. Within 30 min to 2 hrs, the recovery of the sample is nearly quantitative. DNA fragments recovered can be directly subjected to DNA sequence analysis or enzymatic reactions after ethanol precipitation. Proteins can also be recovered after separation by acrylamide gel in the presence or absence of detergents and be ready for further analysis.  相似文献   

15.
Plasmid DNA from Escherichia coli was isolated by electroelution carried out in an agarose gel that contains an incorporated dialysis membrane. As the relative mobility of circular plasmid DNA to linear chromosomal DNA increases when the agarose concentration is decreased, we were able to purify plasmids of up to 50 kbp in 0.3% agarose gel in Tris acetate buffer yielding 10-60 g DNA ml bacterial culture.  相似文献   

16.
The membrane trap is a new device for the electroelution of all kinds of charged macromolecules from gels. Instead of dialysis membranes, the membrane trap uses a new membrane. Retention of macromolecules in an electric field by dialysis membranes depends on the presence of sodium dodecyl sulfate (SDS) in the buffer. The new membrane retains all charged macromolecules larger than approximately 5000 Da without adsorbing them, independent of the use of SDS. Here we report the electroelution of five different lipophilic membrane proteins (33 to 193 kDa) of Mycoplasma pneumoniae from preparative SDS-polyacrylamide gels into a 300-microliter recovery volume. After an 8-h elution period, recovery ranged from 80 (193 kDa) to 97% (33 kDa). The "losses" were generally due to proteins still remaining in the gel slice. All of the eluted proteins tested in a dot-blot assay proved to be antigenically active. The advantages of the device described here are easy handling (insertion of membranes, open system), quantitative recovery, and high reproducibility of the elution results.  相似文献   

17.
Protein recovery from gel electrophoresis plays an important role in functional genomics and proteomics but faces a series of issues (e.g., complex procedure, low recovery, long experimental time). In this study, a monolithic column electroelution (MCE) was developed for protein recovery from gel electrophoresis. With the model proteins of bovine serum albumin (BSA), hemoglobin (Hb), and myoglobin (Mb), the developed device and method were compared with common electroelution procedures in agarose gel electrophoresis (AGE). The comparative experiments revealed that (i) the protein recovery achieved with the developed device was greater than 83%, much higher than the 41% to 50% achieved with the common devices; (ii) the running time to obtain 70% recovery was approximately 15min, evidently shorter than the 240min with the common devices; and (iii) the device and procedure were simple and less time-consuming as compared with those of the common devices. It was observed that the serum protein bands cut from polyacrylamide gel electrophoresis could be transferred into solution in 15 to 30min with 82% yield. The device, along with its relevant procedure, has potential use in protein extraction and proteomics as well as in DNA studies.  相似文献   

18.
以尿激酶的为材料,探索一种从SDS-PAGE胶上回收蛋白 做MALDI-TOF质谱的方法,所用的回收方法包括电洗脱、脱盐、除SDS等过程,电洗脱用的是高盐阻断法,脱盐用的超滤技术,去除SDS用的是冷丙沉淀法,结果证明,此方法至少对一些蛋白质是可行的。  相似文献   

19.
一种SDS-PAGE与MALDI-TOF质谱联用的方法   总被引:2,自引:0,他引:2  
以尿激酶原为材料,探索一种从SDS-PAGE胶上回收蛋白质做MALDI-TOF质谱的方法.所用的回收方法包括电洗脱、脱盐、除SDS等过程.电洗脱用的是高盐阻断法,脱盐用的是超滤技术,去除SDS用的是冷丙酮沉淀法.结果证明,此方法至少对一些蛋白质(如尿激酶原和牛血清白蛋白)是可行的.  相似文献   

20.
We have made a significant improvement in the electroelution device, Elutrap (Schleicher and Schuell) by substituting an agarose gel barrier, which is made from 0.6% agarose (SeaKem GTG; FMC Corporation), into the elution chamber in place of the manufacturer specified BT2 membrane. This modification substantially increases the DNA recovery from agarose gels, even in samples containing less than 1 microgram of DNA, and shortens elution times particularly for large sizes of DNA (greater than 4.4 kbp). Additionally, the gel barrier provides a reproducible quantity and quality of DNA recovery. The high quality of the eluted DNA using the modified Elutrap makes this system suitable for further DNA manipulations.  相似文献   

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