Abstract:
:We describe here a simple and rapid method for enzymatic DNA amplification using DNA template recovered from membrane filters previously used in hybridization analysis. This is done by first solubilizing membrane pieces carrying DNA of interest in dimethyl sulfoxide, followed by isopropanol precipitation and polymerase chain reaction amplification. The source of membrane-bound DNA successfully tested includes plasmid and human leukocyte DNA and DNA immobilized on bacterial colony filters and plaque lifts. The sensitivity of the procedure is such that DNA recovered from 0.5 microgram of filter-bound total human DNA was enough for PCR amplification of a 0.3-kb fragment. Our protocol will be useful for recycling of scarce DNA samples for cloning and sequencing purposes.
journal_name
Biotechniquesjournal_title
BioTechniquesauthors
Chong KY,Chen CM,Choo KBsubject
Has Abstractpub_date
1993-04-01 00:00:00pages
575-8issue
4eissn
0736-6205issn
1940-9818journal_volume
14pub_type
杂志文章相关文献
BIOTECHNIQUES文献大全abstract::A method for the isolation of total cytoplasmic RNA and high molecular weight DNA from the same cells is described. Cells are gently lysed with NP40 in the presence of vanadyl ribonucleoside complex and the nuclei pelleted by centrifugation. RNA is purified by phenol/CHCl3 extraction of the lysate supernatant followed...
journal_title:BioTechniques
pub_type: 杂志文章
doi:
更新日期:1990-02-01 00:00:00
abstract::The Cre/lox system is a powerful genetic tool with which to manipulate the genome. Here, we describe the development of a simple reporter system for Cre recombinase, called the Cre Stoplight. In the absence of Cre, the red fluorescent protein is expressed; when Cre catalyzes a recombination event, the green fluorescen...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/01315st03
更新日期:2001-11-01 00:00:00
abstract::Effective data analysis of the modern biological microscopy data set often necessitates a variety of different analysis strategies, and this often means the biologist may need to use a combination of software tools both commercial and often-times open source. To facilitate this process, there needs to be knowledge of ...
journal_title:BioTechniques
pub_type: 杂志文章,评审
doi:10.2144/000112511
更新日期:2007-07-01 00:00:00
abstract::In this study, we developed a method that allows cDNA library construction from a small amount of RNA without causing serious size bias in the resulting cDNA population. For this purpose, we adopted two-round cRNA amplification by T7 and SP6 RNA polymerases. The first-round cDNAs, flanked by the promoter sequences of ...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/05383RR01
更新日期:2005-03-01 00:00:00
abstract::In this study, we have developed an air-interface culture system in which guinea pig tracheobronchial epithelial (GPTE) cells rapidly form tight monolayers. Enzymatically isolated GPTE cells were plated on collagen-treated polycarbonate microporous cell culture inserts at a density of 10(6) cells/cm2 (day 0). Bioelect...
journal_title:BioTechniques
pub_type: 杂志文章
doi:
更新日期:1993-09-01 00:00:00
abstract::In this report we present a rapid and inexpensive PCR-based method to screen recombinant DNA libraries. The efficiency of this method was demonstrated by the isolation of clones of interest from three different libraries using different vector systems. This method is nonradioactive and makes it easier to handle a larg...
journal_title:BioTechniques
pub_type:
doi:
更新日期:1994-01-01 00:00:00
abstract::Antibodies specific to avian myeloblastosis virus envelope glycoprotein gp80 were raised. Immunoliposomes were prepared using anti-avian myeloblastosis virus envelope glycoprotein gp80 antibody. The antibody was palmitoylated to facilitate its incorporation into lipid bilayers of liposomes. The fluorescence emission s...
journal_title:BioTechniques
pub_type: 杂志文章
doi:
更新日期:1991-08-01 00:00:00
abstract::Haplotypes are useful in population genetics and medicine. Determining haplotypes in the absence of DNA samples from appropriate family members can be difficult and laborious. We have developed a rapid and reproducible method for haplotyping an individual in the absence of relatives. The method utilizes pairs of allel...
journal_title:BioTechniques
pub_type: 杂志文章
doi:
更新日期:1991-04-01 00:00:00
abstract::This paper reports a novel method for the identification of nucleic acid target sequences when these targets have high sequence identity. Homologous genes are currently identified by sequencing. We hypothesize that by primer extension in the presence of selected nucleotides, genes with similar sequence can be identifi...
journal_title:BioTechniques
pub_type:
doi:
更新日期:1993-06-01 00:00:00
abstract::In the quest to move more basic research discoveries into the clinic, the medical community is building training programs to help researchers gain "translational" skills. Sarah Webb explores how PhDs are learning to speak the language of the clinic. ...
journal_title:BioTechniques
pub_type: 新闻
doi:10.2144/000114194
更新日期:2014-08-01 00:00:00
abstract::Changes in cardiac structure that depart from normal have generally been termed "remodeling". Assessment of ventricular remodeling at the cellular level should include measurement of myocyte dimensions. A well-established and reliable method to assess myocyte remodeling uses isolated cells and the Coulter Counter/Chan...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/98253pf02
更新日期:1998-09-01 00:00:00
abstract::Three-dimensional collagen gel contraction is the standard assay utilized for functionally quantifying a variety of cell types, in particular smooth muscle cells (SMCs) and myofibroblasts. Here, we have developed a method to effectively reduce the three-dimensional parameters of the standard collagen gel into a single...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/000113349
更新日期:2010-02-01 00:00:00
abstract::Scanning conductance microscopy investigations were carried out in air on human chromosomes fixed on pre-fabricated SiO2 surfaces with a backgate. The point of the investigation was to estimate the dielectric constant of fixed human chromosomes in order to use it for microfluidic device optimization. The phase shift c...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/000112676
更新日期:2008-02-01 00:00:00
abstract::An assay is described in which an oligonucleotide probe is specifically digested by lambda exonuclease only when it is annealed to its complementary sequence. In this assay, a cycling effect occurs whereby a small amount of target sequence acts as a specific co-factor in the enzymatic degradation of a larger number of...
journal_title:BioTechniques
pub_type: 杂志文章
doi:
更新日期:1992-12-01 00:00:00
abstract::A simple and rapid procedure for recovering the denaturing effect of methylmercuric hydroxide in agarose gel electrophoresis is described. The procedure consisted of the treatment of the commercial methylmercuric hydroxide solutions with Amberlite, a mixture of anion- and cation-exchange resins. This treatment greatly...
journal_title:BioTechniques
pub_type: 杂志文章
doi:
更新日期:1992-04-01 00:00:00
abstract::Planar lipid bilayers represent a versatile platform for studying the functions of various membrane proteins as well as the development of biosensors. Despite the continuing technological progress in the fabrication of low-noise bilayer setups with mechanically and electrically stable planar bilayers, there is still a...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/000112384
更新日期:2007-03-01 00:00:00
abstract::In situ hybridization techniques typically employ chromogenic staining by enzymatic amplification to detect domains of gene expression. We demonstrate the previously unreported near infrared (NIR) fluorescence of the dark purple stain formed from the commonly used chromogens, nitro blue tetrazolium (NBT) and 5-bromo-4...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/000112476
更新日期:2007-06-01 00:00:00
abstract::Human telomerase, a ribonucleoprotein enzyme is known to be associated with immortalized cancer cells but is absent in most normal tissues. Thus, telomerase appears to be an attractive new target for anticancer agents and an important diagnostic marker of human cancers. Here, we describe an improved telomerase assay m...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/98256cr03
更新日期:1998-12-01 00:00:00
abstract::Cell-imaging approaches using new laser-based technologies have a wide applicability to thefields of pathology and cell biology. Here, we present the application of several of these techniques, including confocal scanning laser microscopy (CSLM), laser scanning cytometry (LSC), and laser capture microdissection (LCM),...
journal_title:BioTechniques
pub_type: 杂志文章,评审
doi:10.2144/01314rv01
更新日期:2001-10-01 00:00:00
abstract::Presepsin is a 13-kDa N-terminal glycoprotein of CD14. Previously, agitation-induced increases in presepsin levels have been reported; however, the mechanism remains poorly understood. In this study, we aimed to reveal the mechanism of presepsin increase. The agitated plasma or serum was separated using gel exclusion ...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/btn-2020-0136
更新日期:2021-01-29 00:00:00
abstract::14CO2 capture is commonly used to evaluate the cellular oxidation rate of respiratory substrates. A modification of the established 14CO2-capture method was developed that enables the use of cells in adherent culture and easy analysis of multiple samples under different culture conditions. The use of commercially avai...
journal_title:BioTechniques
pub_type:
doi:10.2144/98245st04
更新日期:1998-05-01 00:00:00
abstract::In the design of oligonucleotide sequences for targeting DNA or RNA sequences, it can be difficult to identify sequences that will hybridize only to the intended target. The term "sequence-specific" or "sequence-nonspecific" is often used to describe the interactions of an oligonucleotide with a mixture of DNA or RNA....
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/96206pf01
更新日期:1996-06-01 00:00:00
abstract::An improved Huffman coding method for information storage in DNA is described. The method entails the utilization of modified unambiguous base assignment that enables efficient coding of characters. A plasmid-based library with efficient and reliable information retrieval and assembly with uniquely designed primers is...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/000113218
更新日期:2009-09-01 00:00:00
abstract::We have designed and built a magnetic tweezers device that enables the application of calibrated stresses to soft materials while simultaneously measuring their microscale deformation using confocal microscopy. Unlike previous magnetic tweezers designs, our device is entirely portable, allowing easy use on microscopes...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/000113701
更新日期:2011-07-01 00:00:00
abstract::We describe a simple and efficient technique that facilitates the amplification of specific mRNA for cloning and sequencing purposes. An mRNA bound to a small piece of membrane filter is used as a template to synthesize complementary DNA. The product of this reaction is then transferred to a new tube and amplified usi...
journal_title:BioTechniques
pub_type:
doi:
更新日期:1993-11-01 00:00:00
abstract::Ribonucleases H (RNases H) are enzymes that specifically degrade the RNA of RNA-DNA hybrids. These enzymes are involved in DNA replication, reverse transcription (RT) and antisense oligodeoxyribonucleotide-mediated arrest of translation. One of the most valuable tools for assaying RNase H activity is the renaturation ...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/97235rr01
更新日期:1997-11-01 00:00:00
abstract::A direct method to study essential genes is to construct conditional knock-down mutants by replacement of their native promoter by an inducible one. In Mycobacteria, replacement of an essential gene promoter with an anhydrotetracycline inducible one was successfully used but required a multi-step approach. In this wor...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/btn-2018-0074
更新日期:2018-09-01 00:00:00
abstract::The use of affinity-based tools has become invaluable as a platform for basic research and in the development of drugs and diagnostics. Applications include affinity chromatography and affinity tag fusions for efficient purification of proteins as well as methods to probe the protein network interactions on a whole-pr...
journal_title:BioTechniques
pub_type: 杂志文章,评审
doi:10.2144/000112803
更新日期:2008-04-01 00:00:00
abstract::Integral membrane G protein-coupled receptors (GPCRs) compose the single most prolific class of drug targets, yet significant functional and structural questions remain unanswered for this superfamily. A primary reason for this gap in understanding arises from the difficulty of forming soluble, monodisperse receptor m...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/000112169
更新日期:2006-05-01 00:00:00
abstract::Few studies have compared the quantification of mRNA by DNA microarray to the results obtained by reverse transcription PCR (RT-PCR). In this study, mRNA was collected from the healing femoral fracture callus of adult and juvenile rats at various times after fracture. Ten samples were measured by both methods for 26 g...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/04364ST02
更新日期:2004-04-01 00:00:00