首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到10条相似文献,搜索用时 135 毫秒
1.
A method for the reproducible and quantitative electrotransfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to a single sheet of either Zetabind or Gene Screen Plus membranes is presented. This procedure uses commercially available equipment and includes three crucial parameters: the omission of methanol from the transfer buffer, the use of thin (0.75-mm) resolving gels, and a newly developed protocol for pretreatment of the polyacrylamide gel after electrophoresis and before electroblotting. This combination of parameters yields a blot that both qualitatively and quantitatively reflects the proteins in the original polyacrylamide gel.  相似文献   

2.
One-hour downward alkaline capillary transfer for blotting of DNA and RNA.   总被引:35,自引:0,他引:35  
The downward alkaline capillary transfer of DNA and RNA from agarose gel to a hybridization membrane was performed using a transfer solution containing 3 M NaCl and 8 mM NaOH. Under mild alkaline conditions, DNA and RNA were completely eluted from the agarose gel and bound to a hybridization membrane within 1 h. On the basis of this new method of transfer a blotting protocol, downward alkaline blotting, was elaborated. It provides a fast and efficient alternative to commonly used Southern and Northern blotting protocols. The downward alkaline blotting of DNA and RNA can be completed in 2.5 and 1.5 h, respectively, and can be used with both plastic and nitrocellulose membranes. In addition, the downward alkaline blotting protocol allows for a hybridization efficiency of DNA and RNA higher than that of the standard blotting protocols performed at neutral pH.  相似文献   

3.
H Saluz  J P Jost 《Gene》1986,42(2):151-157
Some critical parameters of genomic sequencing are described. As an example we show a 200-nucleotide (nt) sequence of the estradiol-regulated avian vitellogenin gene II upstream region containing four CpG nt pairs. The two CpG's at positions -612 and -618 are in the sequence binding estradiol-receptor complex. While all four CpG's are methylated in erythrocytes, they are hypomethylated in the DNA of estradiol-responsive organs of egg-laying hens. A simple electroblot DNA transfer system which gives no distortion of DNA bands and quantitative transfer of denatured DNA from the gel to the filter membrane is described. Using Gene Screen membranes, a maximal hybridization signal was obtained when 30-50% of the input DNA was stably bound to the filters. Hybridization background signal and exposure time could be largely reduced by using highly purified fractionated DNA. Using a 90-120 nt long homogeneous single-stranded DNA probe of high specific activity it was possible to read a genomic sequence of up to 200 nt. The resolution was further improved by reducing the extent of chemical modifications of the DNA during the Maxam-Gilbert sequencing reactions.  相似文献   

4.
The rapid alkaline transfer of high molecular weight DNA from agarose gels to nylon membranes has greatly decreased the time required for setup of Southern transfers. This technique has been used to resolve genomic DNA greater than 1000 base pairs by conventional electrophoresis on 1% agarose gels followed by alkaline transfer to nylon membrane. Now we report that this rapid alkaline method can be used for the transfer of low molecular weight DNA fragments (10 to 1000 base pairs) from NuSieve GTG agarose gels to nylon membrane.  相似文献   

5.
A simple and efficient transfection technique based on lipopolyamine-coated DNA that can be used for gene transfer in cerebellar granular neurons is described. Gene transfer is achieved by exposure of cells to a DNA/lipid complex obtained by simple mixing of lipopolyamine and plasmid DNA. This procedure may represent a general tool of physiological investigations in primary cells. We show that the promoters of the introduced chimera genes are regulated by their respective trans-acting factors and may be modulated via membrane receptors and second messengers. This procedure has no noticeable toxic effects, nor does it seem to interfere with complex physiological behavior like neuronal differentiation.  相似文献   

6.
The alkaline elution technique has been modified to be used in the isolation of DNA replication intermediates and in the study of the process of DNA replication. In this procedure pulse labeled CHO cells are layered onto a membrane filter, lysed with detergent, and the nascent DNA eluted in step-wise fashion with tetrapropylammonium hydroxide at pH 11.0, 11.3, 11.5 and 12.1. Alkaline sucrose sedimentation of the eluted DNA shows that the pH 11.0 material consists of < 9S fragments consistant with those described by Okazaki and others. DNA eluting at pH 11.3 has a molecular weight of 8–12 million daltons, DNA which elutes at pH 11.5 sediments with a molecular weight of 20–30 million daltons. Two independent lines of evidence suggest that the pH 11.3 material includes DNA sequences synthesized at replicon origins. (1) Exposure of cells to low doses of X-ray prior to pulse labeling reduces the pH 11.3 fraction by 40–50% while there is little change in the other fractions. (2) Synchronization of cells by inhibiting DNA synthesis with FdU, followed by a 2 min pulse label, yields approximately 50% of the incorporated 3H-thymidine in the pH 11.3 fraction. The pH step elution technique has the following advantages: 1. Intermediates of high specific activity can be isolated from 106 cells per filter; 2. By lysing cells on a filter, proteins, nucleases, and other cellular materials are eliminated; 3. DNA in the lysate is never handled, thus eliminating shearing; 4. Eluted DNA may be instantaneously neutralized by collecting into a buffer to protect it from alkaline degradation.  相似文献   

7.
8.
To study the mechanisms of formation and repair of DNA-protein crosslinks in mammalian cells, the best general method to assay these lesions is the Kohn membrane alkaline elution procedure. Use of this sensitive technique requires the introduction of random strand breaks in the DNA by X-irradiation to reduce the very high molecular weight so that it elutes off the filter at an appropriate rate. This report describes an alternative method for fragmenting the DNA in the absence of X-irradiation equipment. Convenient reproducible elution rates of DNA from various mouse and human cells in culture without X-irradiation result from elution through polyvinyl chloride filters with 75 mM sodium hydroxide (0.33 ml/min) instead of the standard 20 mM EDTA-tetrapropylammonium hydroxide, pH 12.2 (0.03 to 0.04 ml/min). Dose-dependent retardation of the DNA elution was observed over the range 0 to 30 microM trans-platinum(II)diamminedichloride, and proteinase K treatment during cell lysis restored the elution rate to that of the untreated control cell DNA. In the absence of X-irradiation, this elution method measures DNA-protein crosslinks with higher sensitivity and equivalent reproducibility as the air-burst procedure.  相似文献   

9.
Novel reduced sugar gemini amphiphiles linked through their tertiary amino head groups via alkyl spacers of 4 or 6 carbons, and with varying (unsaturated) alkyl tail lengths of 12--18, have been synthesized and tested for transfection in vitro in an adherent Chinese hamster ovary cell line (CHO-K1). Transfection efficiencies peaked at 2.7 times that of the commercial standard Lipofectamine Plus/2000 for pure solutions of the compound bearing unsaturated (oleyl) alkyl tails. For those compounds bearing saturated alkyl tails, transfection efficiency peaked at a tail length of 16, at a level similar to Lipofectamine Plus/2000. All of the amphiphiles formed bilayer vesicles at physiological pH. Some of the amino groups at the surface were protonated, and vesicles therefore bore a positive charge. Increased protonation with reduced pH resulted in greatly increased monomer solubility and a morphology change from vesicle to micelle at characteristic pH values, dependent on the tail length. For the compounds promoting high transfection efficiency, this characteristic pH was within the range found in the endosomal compartment (7.4--4.0). Formation of mixed micelles between gemini surfactant and membrane phospholipids at reduced pH may therefore provide a method of endosome rupture and subsequent escape of entrapped DNA, thus discarding the need for extra fusogenic or endosomolytic agents. The positive charge on the vesicles at physiological pH drives the colloidal association with DNA. Small angle X-ray scattering measurements indicate that lamellar aggregates are formed, which have a d spacing of 48--54 A. Preliminary differential scanning calorimetric measurements suggest that reduction of pH causes a disordering of the hydrocarbon region of the DNA-surfactant complex.  相似文献   

10.
A rapid alkaline extraction procedure for screening recombinant plasmid DNA.   总被引:3200,自引:408,他引:2792  
A procedure for extracting plasmid DNA from bacterial cells is described. The method is simple enough to permit the analysis by gel electrophoresis of 100 or more clones per day yet yields plasmid DNA which is pure enough to be digestible by restriction enzymes. The principle of the method is selective alkaline denaturation of high molecular weight chromosomal DNA while covalently closed circular DNA remains double-stranded. Adequate pH control is accomplished without using a pH meter. Upon neutralization, chromosomal DNA renatures to form an insoluble clot, leaving plasmid DNA in the supernatant. Large and small plasmid DNAs have been extracted by this method.  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号