共查询到18条相似文献,搜索用时 78 毫秒
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《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
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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
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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
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目前在国内分子生物学实验室所采用的多种从琼脂糖凝胶上回收DNA的方法普遍存在着各种问题。本文介绍一种新的从琼脂糖凝胶上分离和提取DNA简易装置。实验表明用这种装置回收DNA具有高效、快捷和经济的优点,非常适合在国内实验室普及推广。 相似文献
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DNA印迹(Southern blot)杂交法是研究DNA分子结构,变异及其组成的一种分子生物学技术。自1975年闻世以来,在分子生物学,遗传学及分子病毒学等研究领域得到广泛应用,对这些学科的发展起了重要作用。由于该技术均需要首先将电泳后已变性的DNA从琼脂糖凝胶转移至支持膜上,因此,实验结果的好坏很大程度取决于吸印的效果,同时也受支持物 相似文献
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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
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We demonstrate a novel method for stretching a long DNA molecule in agarose gel with alternating current (AC) electric fields. The molecular motion of a long DNA (T4 DNA; 165.6 kb) in agarose gel was studied using fluorescence microscopy. The effects of a wide range of field frequencies, field strengths, and gel concentrations were investigated. Stretching was only observed in the AC field when a frequency of ∼10 Hz was used. The maximal length of the stretched DNA had the longest value when a field strength of 200 to 400 V/cm was used. Stretching was not sensitive to a range of agarose gel concentrations from 0.5 to 3%. Together, these experiments indicate that the optimal conditions for stretching long DNA in an AC electric field are a frequency of 10 Hz with a field strength of 200 V/cm and a gel concentration of 1% agarose. Using these conditions, we were able to successfully stretch Saccharomyces cerevisiae chromosomal DNA molecules (225-2,200 kb). These results may aid in the development of a novel method to stretch much longer DNA, such as human chromosomal DNA, and may contribute to the analysis of a single chromosomal DNA from a single cell. 相似文献
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目的:介绍了一种从普通琼脂糖电泳中回收DNA的简便、快捷、高效且廉价的方法.方法:利用0.5 mL离心管、1.5mL离心管、尼龙膜做成的一个小装置.把含有DNA的凝胶放在膜上,离心,收集从管底流出来的液体,用乙醇沉淀DNA.结果:最终回收率为60%左右,回收率大约为市售试剂盒的90%,接近市售DNA回收试剂盒.结论:该方法操作简单,回收率高,无其他试剂污染. 相似文献
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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
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用琼脂糖凝胶电泳法同时测定肌酸激酶(CK)同工酶及其亚型(CK-MB亚型、CK-MM亚型),其特点是以不连续缓冲液体系为基础,在较低电压条件下,30min完成分离过程,能同时报告CK-MB同工酶,CK-MB1,CK-MB2以及CK-MM1,CK-MM2,CK-MM3的百分含量.CK-MB和CK-MM同工酶的亚型最低检出限分别为2.2U/L,3.2U/L.该法具有快速、灵敏度高、重复性好、无需特殊仪器、操作简单等优点. 相似文献
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The polymorphic transferrin picture in the sera from 894 Swedish cattle was investigated with an agarose gel electrophoresis technique. The serum transferrin bands in the electrophoresis pattern were first identified by labelling with 59Fe. Six existing phenotypes based on the alleles TfA, TfD and TfE could be detected. The frequencies of transferrin types and transferrin alleles are presented, and it is concluded that there are great differences in the frequencies between the Swedish Red and White and the Swedish Friesian. 相似文献