从琼脂糖凝胶中高效回收DNA技术的探讨

用两只离心管制成的凝胶过滤装置,从电泳后的琼脂糖凝胶中回收DNA片段的简易方法。它依次包括以下步骤：凝胶过滤装置的制作、凝胶切割、凝胶低温冷冻、低温高速离心、ddH20洗胶、DNA纯化和回收效果检测等。用此方法回收的DNA片段产率高、质量纯,可直接用于分子生物学实验的后续操作,如载体连接、PCR模板获得、DNA探针制备、基因测序等。其优点是：DNA片段的回收率高（90％以上）,质量好;操作简便,耗时短;回收装置简单,成本低廉,可进行商品化开发。     

琼脂糖凝胶中DNA片段的挤压回收法

为了简化琼脂糖凝胶中DNA片段的回收,该文报道一种新的挤压回收法。将含有DNA片段的凝胶块放在折叠的封口膜之间,然后用一小塑料平板将凝胶块中的DNA溶液用力挤出,用移液枪把所有挤压出的DNA溶液放入effendorf管中,然后用常规的苯酚抽提法进行纯化。DNA回收率达到40-60%(w/w)。该方法简单而有效,且回收的DNA能够直接应用于酶切、连接及PCR等各种分子生物学操作。     

一种用于PCR模板制备的电泳产物简易回收方法

为了探索一种简便、有效而且能从琼脂糖凝胶中大量回收用于第2次PCR扩增的DNA电泳条带的方法,采用刀片切胶法和牙签插胶法从琼脂糖中回收DNA,并进行了两种方法的比较．结果显示牙签插胶法回收的DNA用作第2次PCR的模板,获得了清晰、稳定的PCR产物电泳条带,用该法成功地制备了一批DNA微阵列探针．由此可见牙签插胶法是一种简便、快速、有效的用于PCR模板的DNA琼脂糖凝胶回收法．     

一种有效回收小分子DNA片段的方法

介绍了一种有效回收小于200bp DNA片段的方法。用改进的冻融-离心回收法对155bp的DNA片段进行回收,并且与常规冻融-离心回收、TaKaRa回收试剂盒的结果做对比,用琼脂糖凝胶电泳检测回收结果,紫外吸收法定量分析。结果证明:改进的方法是一种经济、方便、可靠的回收小分子DNA片段的方法。     

一种从凝胶简单快速回收核酸的好方法

从凝胶中分离DNA或DNA片断是基因工程中常用的手段.在重组DNA、分子杂交、基因诊断等实验中,经常需要从凝胶中分离并回收核酸或核酸片断.目前分离DNA片断的方法很多.常用的如电洗脱法、冻融法等操作烦琐且得率较低;低融点琼(王旨)糖法得率较高,但价格昂贵,特别是回收的DNA片断常混有细小琼(王旨)糖颗粒,影响以后的酶切、连接等.我们摸索了一种简便方法,能够从琼(王旨)糖凝胶中高纯度、高得率地分离核酸或核酸片断,回收所得的DNA片断可以直接用于各种酶切反应和基因重组实验.现以分离P~(ZB24)重组质粒中2.3kb的大R蠖核多角体基因片断为例,说明如下:     

从非变性聚丙烯酰胺凝胶回收纯化DNA样本

介绍一种从非变性聚丙烯酰胺凝胶中回收和纯化目标DNA片段的方法,经比较回收纯化前后PCR产物的电泳结果,表明该方法具有简单快捷和效率高的优点。     

一种从琼脂糖凝胶中同步回收DNA和琼脂糖的方法

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。     

回收琼脂糖凝胶中DNA的简便方法

多年来国内分子生物学实验室所采用的几种从琼脂糖凝胶中回收DNA的方法不同程度地存在着各种问题。现介绍一种新的从琼脂糖凝胶上分离和提取DNA的简易方法,采用实验室常用的吸附柱中的吸附膜对DNA进行拦截、纯化和回收。实验证明这种方法回收DNA具有简便快捷、成本低和效率高等优点,是一种切实可行的方法。     

一种简单快速回收DNA片段的方法——DE-81滤纸

琼脂糖凝胶电泳对于快速分离不同分子量的DNA片段是极为有效的,但把DNA片段从凝胶中回收回来有时要碰到一些困难,如回收的DNA易受凝胶中硫酸多糖的污染(该成分是许多酶的抑制剂,如内切酶、连接酶、激酶、聚合酶等);大片段DNA回收     

利用机油提高琼脂糖凝胶中DNA片段的回收率

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.     

Membrane cofactor protein of the complement system. A HindIII restriction fragment length polymorphism that correlates with the expression polymorphism.

An RFLP was found in the DNA of 25 unrelated persons, two families, and five cell lines that correlated with their membrane cofactor protein phenotype. If restricted with HindIII, DNA derived from upper band predominant protein (U) phenotypes had a band at 2 kb, whereas DNA of lower band predominant (L) phenotypes had a 4-kb band. The equal band protein phenotype, in which equal quantities of the two species are expressed, had bands at both 4 and 2 kb. The polymorphic HindIII site was localized to an intron within the membrane cofactor protein gene between exon 1 (codes for 5'UT/signal peptide) and exon 2 (codes for the first short consensus repeat). Using the polymerase chain reaction (PCR), sequences around this site were amplified and a single band of 260 bp was produced. In the U phenotype, the PCR product was restricted with HindIII into 200- and 60-bp fragments. In the L phenotype, there was no change in the size of 260 bp upon restriction with HindIII. For the equal band protein phenotype, the PCR product was partially cleaved. The 260-bp PCR product was subcloned and sequenced. DNA from the U phenotype demonstrated an intact HindIII site (AAGCTT), whereas in the DNA of the L phenotype, this site was altered because a "G" was substituted for a "C" (AAGGTT).     

The interaction of calmodulin with erythrocyte membrane proteins

The method of sedimentation equilibrium in an air-driven ultracentrifuge (Airfuge) has been employed to investigate the interaction of 125I-calmodulin with the cytoskeletal components of the human red cell membrane. The results indicate significant calcium-dependent calmodulin binding activity in the low and high ionic strength extracts of the human erythrocyte membrane. The interaction of 125I-calmodulin with the low ionic strength extract proteins is analysed quantitatively. Further purification of the high ionic strength extract comprising mainly band 2.1 and band 4.1 results in the elution of calmodulin binding activity in a purified fraction of band 4.1.     

Ordered replication of DNA sequences: synthesis of mouse satellite and adjacent main band sequences.

The replication of mouse satellite DNA was delayed when synchronized 3T3 cells were exposed to low concentrations of hydroxyurea during S phase, It appears that the onset of satellite replication is not a time dependent event, but instead requires that a certain amount of main band DNA be synthesized first. Using hydroxyapatite chromatography and S1 nuclease digestion, a procedure was developed to quantitate the synthesis of both satellite and neighboring main band sequences. The replication kinetics of satellite determined by this method agree with previous estimates. Main band sequences adjacent to satellite appear to replicate in concert with satellite DNA. The results are discussed and related to the limitations of the techniques utilized.     

Membrane attachment of replicating parental DNA molecules of bacteriophage M13

Early in the infection with bacteriophage M13 the infecting parental DNA strand becomes attached to the host cell membrane. Using a gentle lysis procedure followed by sucrose gradient centrifugation, up to 80% of the parental DNA co-sediment with the bacterial membranes. The membrane fraction was deproteinized by phenol extraction and the solubilized DNA was further analysed by band sedimentation in neutral and alkaline CsCl gradients. Between 5′ and 15′ after infection at least half of the membrane bound parental DNA was found to be incorporated into replicative intermediates with viral strands of more than unit length.     

Isotachophoresis of nucleic acids in agarose gel rods

A new method of electrophoresis (isotachophoresis in agarose gel rods) in which nucleic acid molecules are not separated but, oppositely, are brought together into one band, was elaborated. Heterogeneous in size DNA and RNA polymers present in a few milliliters of a solution at so low concentration that their isolation by other methods is hardly attainable and fraught with losses are brought together into one visible narrow band when put in a discontinuous electric field. Polynucleotides migrate in dilute (0.1%) semifluid agarose gel that permits easy quantitative isolation of the band of interest. Resulting DNA can be used directly in PCR. The suggested method for isolation of micro amounts of nucleic acids from dilute solutions can be applied to forensic and clinical research and cancer gene diagnostics by the analysis of fragmented circulating DNA from bodily fluids.     

Freeze-fracture study of the site of attachment of Cryptosporidium muris in gastric glands.

The mode and organization of the attachment site of Cryptosporidium muris to gastric glands of stomach were investigated by the freeze-fracture method. Cryptosporidium muris was enveloped by a double membrane, of host plasma membrane origin, which formed the parasitophorous vacuole. The outer membrane of the double membrane was continuous with host plasma membrane, while the inner membrane was connected with the anterior part of the parasite plasma membrane at the annular ring. The density of intramembranous particles (IMP) was severely altered at the above two junctures. The parasitophorous outer membrane showed low IMP-density when compared to the host plasma membrane, although both membranes were continuous at the dense band. The inner membrane had few IMP, whereas the parasite plasma membrane showed numerous IMP, although both membranes were continuous at the annular ring. The size of dense band and annular ring was similar in diameter. The feeder organelle was clearly visible as membrane folds in freeze-fracture and some of them were connected with small vesicles of cytoplasm, indicating that the feeder organelle may play an important role for incorporation of nutrients from the host cell.     

Direct amino acid analysis of proteins electroblotted onto polyvinylidene difluoride membrane from sodium dodecyl sulfate-polyacrylamide gel

The band of appropriate proteins (basic pancreatic trypsin inhibitor, soybean trypsin inhibitor, interleukin 2, and human leukocyte interferon alpha A) on a polyvinylidene difluoride (PVDF) membrane, which was electroblotted from sodium dodecyl sulfate (SDS)-polyacrylamide gel and then stained with Coomassie blue R-250, was cut out and directly hydrolyzed in HCl in the presence of thioglycolic acid for amino acid analysis. The analytical values agreed with those expected with recoveries of 29-47%, except that the value for tryptophan was very low or scarcely detected. This method was applied to the identification of human growth hormone (hGH) in a partially purified preparation. The amino acid composition of the band corresponding to about 2 micrograms of hGH agreed with the theoretical values. These results indicate that the band on the PVDF membrane can be directly hydrolyzed for amino acid analysis and that the method can be used for partially purified proteins separated using SDS-polyacrylamide gel electrophoresis.     

Freeze-Fracture Study of the Site of Attachment of Cryptosporidium muris in Gastric Glands

ABSTRACT The mode and organization of the attachment site of Cryptosporidium muris to gastric glands of stomach were investigated by the freeze-fracture method. Cryptosporidium muris was enveloped by a double membrane, of host plasma membrane origin, which formed the parasitophorous vacuole. The outer membrane of the double membrane was continuous with host plasma membrane, while the inner membrane was connected with the anterior part of the parasite plasma membrane at the annular ring. The density of intramembranous particles (IMP) was severely altered at the above two junctures. The parasitophorous outer membrane showed low IMP-density when compared to the host plasma membrane, although both membranes were continuous at the dense band. The inner membrane had few IMP, whereas the parasite plasma membrane showed numerous IMP, although both membranes were continuous at the annular ring. The size of dense band and annular ring was similar in diameter. The feeder organelle was clearly visible as membrane folds in freeze-fracture and some of them were connected with small vesicles of cytoplasm, indicating that the feeder organelle may play an important role for incorporation of nutrients from the host cell.     

Association of the Bacillus subtilis Chromosome with the Cell Membrane: Resolution of Free and Bound Deoxyribonucleic Acid on Renografin Gradients

Linear density gradients of Renografin have resolved two components of bacterial deoxyribonucleic acid (DNA) in sheared lysates. Component 1, at equilibrium density after 5 hr of centrifugation, is enriched for newly synthesized DNA and markers near the origin and terminus of replication. It contains 5% of total cellular protein, 25% of the phospholipids, 30 to 50% of the DNA, 4 to 11% of unstable ribonucleic acid (RNA), RNA polymerase, and low amounts of DNA polymerase. The material is sensitive to Pronase and Sarkosyl. In unsheared lysates, all of the DNA forms a band at this position. Shearing the lysate generates a slow-sedimenting fraction of DNA (component 2) which contains more uniformly labeled than newly synthesized DNA. These observations suggest that replicating DNA and DNA at the origin and possibly the terminus of replication are associated with membrane. The amount of uniformly labeled DNA in component 1 and an estimate of the number of chromosomal fragments suggest that other parts of the chromosome are possibly associated with the membrane.     

Specific protein-DNA complexes: immunodetection of the protein component after gel electrophoresis and Western blotting

A method is described to determine the presence and the relative amount of proteins within specific protein-DNA complexes. The system studied is the LexA repressor from Escherichia coli and its interaction with the operator of the caa gene encoding the bacterial toxin colicin A. After separation of the free and the complexed 32P-labeled DNA on a native polyacrylamide gel, the bound proteins are transferred on a polyvinylidine difluoride (PVDF) membrane after sodium dodecyl sulfate denaturation. Development of the protein on the membrane was achieved on reaction with an anti-LexA antibody and the use of a second anti-antibody crosslinked with alkaline phosphatase. The phosphatase activity is monitored using 5-bromo-4-chloro-3-indolyl phosphate as a substrate and 4-nitroblue tetrazolium salt. A quantitation by densitometry of both the stained protein bands on the PVDF membrane and the DNA on autoradiograms allowed us to assign the relative stoichiometry of the two different complexes formed between LexA and the caa operator. The method should allow unraveling of complicated band shift patterns arising from the presence of several binding sites for a same protein, as in our case, or from the presence of different proteins binding to a same DNA fragment.