共查询到19条相似文献,搜索用时 468 毫秒
1.
2.
3.
目的:发展一种简单快速经济的回收琼脂糖凝胶中DNA的方法.方法:将切的琼脂糖凝胶胶块放入嵌套Eppendorf管中捣碎,加入50μL机油,室温下12 000r/min离心5min,取大Eppendorf管中收集的液体跑胶检验,凝皎上包括代表100bp~2000bp不同大小DNA分子回收率的分子量标准DL2000.结果:DNA回收率可由加机油前的约35%提高为加机油后的45%-90%,回收效率的波动主要取决于DNA片段的大小、切下的含有DNA的胶块的大小及操作者的熟练程度.结论:该方法快速、简便、经济,具有良好的重复性与特异性,比许多国产的试剂盒更可靠. 相似文献
4.
5.
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
6.
一种有效回收短片段PCR产物的方法 总被引:3,自引:1,他引:2
目的:为了有效地回收小于200bp的短片段PCR产物。方法:研究了在-20℃条件下,用无水乙醇和3mol/l醋酸钠共沉淀短片段的PCR产物的方法。结果和结论:用这种共沉淀PCR产物的方法,它能够有效地回收小于200bp以下的DNA片段,并且回收到的片段能够有效的进行酶切反应和T-载体连接反应。这种方法也能够回收酶切后的短DNA片段,并对后来的连接反应没有影响。这种共沉淀回收短片段DNA方法相对于其他方法来不仅具有可行性,而且有经济和操作简单的优点。 相似文献
7.
PCR特异产物回收纯化方法的比较 总被引:1,自引:1,他引:0
方法:采用三种方法对苹果褪绿叶斑病毒RT-PCR的特异DNA产物进行回收纯化。目的:针对不同情况,选择适宜的回收纯化方法。结果:用普通琼脂糖替代低融点琼脂糖,回收纯化后产物的浓度及纯度与低融点琼脂糖法基本一致,完全可以用普通琼脂糖替代低融点琼脂糖进行DNA片段的回收纯化,从而降低成本,简化操作。玻璃奶法的回收纯度明显高于低融点琼脂糖法和普通琼脂糖法,且更快速安全,是采用普通琼脂糖法还是采用玻璃奶法回收纯化DAN片段应以实际需要而定。 相似文献
8.
《生命科学研究》2015,(4):299-302
介绍一种从琼脂糖凝胶同步回收DNA和琼脂糖的方法。利用0.25 mol/L异硫氰酸胍溶液(p H 8.0)溶解含有目的 DNA片段的的凝胶条,胶条溶解后,静置冰上10 min再加入预冷的异丙醇,琼脂糖呈颗粒状析出,通过离心即可初步分离DNA和琼脂糖。上清液用异丙醇沉淀回收DNA片段,利用50%PEG溶液沉淀琼脂糖。分别对0.2 kb、1 kb和10 kb长度的DNA片段进行回收,回收率分别为19.44%、36.40%、13.49%,回收的DNA纯度高,电泳条带清晰。琼脂糖均回收率为62.52%,回收琼脂糖脱水后的状态为白色颗粒。该方法切实可行,回收成本低廉,回收的DNA和琼脂糖可用于后续实验。 相似文献
9.
为筛选和优化出较适宜的苎麻脱胶菌群DNA提取方法,本文分别以来自苎麻沤麻环境的6种纯培养菌等丰度混合物和苎麻自然沤麻菌群为材料,研究"溶菌酶-SDS"法、"超声波-溶菌酶-SDS"法、"蛋白酶K-SDS"法以及"冻融-蛋白酶K-SDS"法4种DNA提取方法对菌群16Sr DNA基因PCR-DGGE偏移结果的影响。结果表明,4种方法均能从2类材料中提取出了超过1600ng/μL的DNA,不同方法之间DNA产率略有差异,经过超声波处理或反复冻融处理的DNA有明显的降解,但4种方法提供的DNA模板均扩增出了450bp的16Sr DNA基因片段。4种DNA提取方法对DGGE结果有明显影响,且只有"冻融-蛋白酶K-SDS"法检测到了纯培养菌混合物中的全部6种细菌,4种方法获得的自然沤麻菌群的DGGE指纹图谱也明显不同,最多产生17条带("冻融-蛋白酶K-SDS"法),最少只有9条带("溶菌酶-SDS"法),增加超声波或冻融等物理处理可以使部分弱带变强。因此,组合应用物理、生物和化学等细胞裂解方法可以提供更有代表性的DNA,可减少PCR-DGGE结果的偏移。 相似文献
10.
11.
A simple, sensitive, dried gel DNA hybridization method for detection of Listeria monocytogenes DNA fragments is described. DNA samples were fractionated on an agarose gel. The gel was then denatured in NaOH-NaCl and neutralized in Tris-NaCl. The resulting agarose gel was dried and hybridized with 32P-labelled DNA probe. No transfer to nitrocellulose membranes was used. 相似文献
12.
It has been shown that minor differences, such as single-base-pair substitutions between otherwise identical DNA fragments can result in altered melting behavior detectable by denaturing gradient gel electrophoresis (DGGE). Sequence variations in only a small DNA region within one locus can be detected using the previously described procedures. We have developed a method for the efficient Southern transfer of genomic DNA fragments from the denaturing gradient gels in order to be able to analyze larger regions in several loci for variation. The gels were made using polyacrylamide containing 2% low-geling-temperature agarose (LGT). The polyacrylamide gel (PAG) was crosslinked with a reversible crosslinker, and after electrophoresis the crosslinks were cleaved, the structure of the gel being maintained by the agarose. After this treatment of the denaturing gels, more than 90% of the DNA fragments could be transferred to nylon membranes by alkaline transfer, while electroblotting transferred only 10% of the DNA. Hybridization with gene-specific probes was then performed. We have used this technique to identify an RFLP in the COL1A2 gene in a human genomic DNA sample. The transfer technique described should make the use of DGGE more widely applicable since the genomic DNA fragments separated on one gel can be screened with several different probes, both cDNA and genomic probes. 相似文献
13.
W G Stroop 《Analytical biochemistry》1988,169(1):194-196
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.
Purification of DNA fragments from acrylamide or agarose gels is a commonly used technique in the molecular biology laboratory.
This article describes a rapid, efficient, and inexpensive method of purifying DNA fractions from an agarose gel. The purified
DNA is suitable for use in a wide range of applications including ligation using DNA ligase. The procedure uses standard high-melting-temperature
agarose and normal TBE electrophoresis buffer. In addition, the protocol does not involve the use of highly toxic organic
solvents such as phenol. 相似文献
15.
J C Bearden 《Gene》1979,6(3):221-234
A new theoretical model for the migration of high-molecular-weight, double-stranded DNA on agarose gels is presented. This leads to the prediction that under certain conditions of electrophoresis, a linear relationship will exist between the molecular weight of a DNA molecule, raised to the (-2/3) power, and its electrophoretic mobility. Agarose gel electrophoresis of the fragments of bacteriophage lambda DNA produced by several restriction endonucleases confirms this relationship, and establishes some of the limits on its linearity. For this work, a polyacrylamide slab gel apparatus was modified for use with agarose gels. This apparatus has several advantages over others commercially available for agarose gel electrophoresis, including the abilities to run a larger number of samples at one time, to use lower-concentration gels, and to maintain better temperature stability across the width of the gel. The validation of the relationship developed here between molecular weight and electrophoretic mobility should make this a useful method for determining the molecular weights of DNA fragments. 相似文献
16.
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
17.
18.
We have synthesized and characterized a thermoreversible hydrogel of multiplied block copolymers, composed of poly(ethylene oxide) and poly(propylene oxide), for DNA electrophoresis. The aqueous solution of block copolymers turned into a hydrogel upon heating at temperatures above 10-11 degrees C, whereas it reverted into a solution upon cooling. Linear double-stranded DNA molecules migrated through the gel matrices at a rate that was inversely proportional to the logarithm of the DNA length. The hydrogel is most effective for separating DNA fragments in the 10- to 2000-bp range. The resolving range lay in-between the effective ranges of polyacrylamide and agarose gel electrophoreses of DNA. The gel slices containing DNA fragments were liquefied by cooling on ice, and the DNA was precipitated with ethanol. No contaminants that inhibit enzymatic reactions were found in the DNA recovered from the hydrogel. Plasmid DNA recovered from the hydrogel was recircularized with T4 DNA ligase and yielded highly efficient Escherichia coli transformation. Therefore, thermoreversible gel electrophoresis will be a useful method for DNA separation and isolation in recombinant DNA technology. 相似文献
19.
Pei Yun Lee John Costumbrado Chih-Yuan Hsu Yong Hoon Kim 《Journal of visualized experiments : JoVE》2012,(62)
Agarose gel electrophoresis is the most effective way of separating DNA fragments of varying sizes ranging from 100 bp to 25 kb1. Agarose is isolated from the seaweed genera Gelidium and Gracilaria, and consists of repeated agarobiose (L- and D-galactose) subunits2. During gelation, agarose polymers associate non-covalently and form a network of bundles whose pore sizes determine a gel''s molecular sieving properties. The use of agarose gel electrophoresis revolutionized the separation of DNA. Prior to the adoption of agarose gels, DNA was primarily separated using sucrose density gradient centrifugation, which only provided an approximation of size. To separate DNA using agarose gel electrophoresis, the DNA is loaded into pre-cast wells in the gel and a current applied. The phosphate backbone of the DNA (and RNA) molecule is negatively charged, therefore when placed in an electric field, DNA fragments will migrate to the positively charged anode. Because DNA has a uniform mass/charge ratio, DNA molecules are separated by size within an agarose gel in a pattern such that the distance traveled is inversely proportional to the log of its molecular weight3. The leading model for DNA movement through an agarose gel is "biased reptation", whereby the leading edge moves forward and pulls the rest of the molecule along4. The rate of migration of a DNA molecule through a gel is determined by the following: 1) size of DNA molecule; 2) agarose concentration; 3) DNA conformation5; 4) voltage applied, 5) presence of ethidium bromide, 6) type of agarose and 7) electrophoresis buffer. After separation, the DNA molecules can be visualized under uv light after staining with an appropriate dye. By following this protocol, students should be able to: 1. Understand the mechanism by which DNA fragments are separated within a gel matrix 2. Understand how conformation of the DNA molecule will determine its mobility through a gel matrix 3. Identify an agarose solution of appropriate concentration for their needs 4. Prepare an agarose gel for electrophoresis of DNA samples 5. Set up the gel electrophoresis apparatus and power supply 6. Select an appropriate voltage for the separation of DNA fragments 7. Understand the mechanism by which ethidium bromide allows for the visualization of DNA bands 8. Determine the sizes of separated DNA fragments 相似文献