Multiple site-directed mutagenesis via simple cloning by prolonged overlap extension.

abstract：:We describe a rapid and sensitive method for the detection of nucleotide sequence variation that can be used for large-scale screening of population markers. Denaturing gradient gel electrophoresis (DGGE) detects sequence variants of amplified fragments by the differences in their melting behavior. DGGE detects most s...

journal_title：BioTechniques

authors： Miller KM,Ming TJ,Schulze AD,Withler RE

更新日期：1999-11-01 00:00:00

abstract：:We have utilized 5-bromo-2'deoxyuridine (BrdU) substituted DNA as a probe for a number of applications including, principally, for chromosome painting by fluorescence in situ hybridization (FISH) but also for DNA end-labeling to detect apoptotic cell death and for filter hybridization. Br-dUTP was used as a substitute...

journal_title：BioTechniques

authors： Mühlmann-Díaz MC,Dullea RG,Bedford JS

更新日期：1996-07-01 00:00:00

abstract：:Thymidine kinase 1 (TK1) is an enzyme involved in DNA precursor synthesis that has been used as a biomarker for prognosis and monitoring of different malignancies. In this study, we compared two immunoassays for measuring TK1 protein concentrations: the TK 210 ELISA (AroCell AB) and TK1 ELISA from Abcam. Overall, the ...

journal_title：BioTechniques

authors： Kumar JK,Holmgren S,Levedahl KH,Höglund M,Venge P,Eriksson S

更新日期：2020-06-01 00:00:00

abstract：:To gain insightful information about the mechanisms through which genes are activated and repressed requires gene reporter systems that are sensitive, robust, and cost-effective. Although numerous reporter gene technologies are commercially available, none are as sophisticated and user-friendly as beta-lactamase (BLA)...

journal_title：BioTechniques

authors： Qureshi SA

更新日期：2007-01-01 00:00:00

abstract：:In this special section of BioTechniques, we examine the role of rapid molecular technologies in the detection and identification of agents of infectious disease (ID) and biological weapons (BWs). Besides the threat posed by the global proliferation of BW technologies, there are numerous emerging and reemerging ID age...

journal_title：BioTechniques

authors： Peruski LF Jr,Peruski AH

更新日期：2003-10-01 00:00:00

abstract：:A magnetic bead-based system for DNA isolation utilizing monodisperse beads was tested with the aim of producing a general approach for PCR-ready DNA. This commercially available system was originally designed for isolating PCR-ready DNA from human whole blood. We tested diverse organisms belonging to the major groups...

journal_title：BioTechniques

authors： Rudi K,Kroken M,Dahlberg OJ,Deggerdal A,Jakobsen KS,Larsen F

更新日期：1997-03-01 00:00:00

abstract：:At many research institutions, lab space is more valuable than gold. Developers are taking note by designing smaller instruments with enhanced capabilities. Nathan Blow looks inside today's tiny lab. ...

journal_title：BioTechniques

authors： Blow NS Ph D

更新日期：2017-01-01 00:00:00

abstract：:Primary human fibroblasts and a series of cell lines (A549, BNL CL.2, H225, NIH 3T3 and Rat-1) are efficiently transfected by using positively charged complexes of plasmid DNA and transferrin-polylysine or polylysine in the presence of glycerol (1 molar to 1.8 molar, depending on the cell type). An increase in gene ex...

journal_title：BioTechniques

authors： Zauner W,Kichler A,Schmidt W,Sinski A,Wagner E

更新日期：1996-05-01 00:00:00

abstract：:Knob heterochromatin served as the model for the development of fluorescent chromosome in situ hybridization on maize meiotic chromosomes. The meiotic chromosomes were hybridized with a digoxigenin-labeled RNA probe of the knob repeat sequence that is a component of the morphologically determined knob heterochromatin....

journal_title：BioTechniques

authors： Makowski ER,Ruzin SE

更新日期：1994-02-01 00:00:00

abstract：:A PCR-based method is described for the production of cDNA libraries and total cDNA probes from a few milligrams of tissue. Using a model system, we show how a PCR library and PCR probes can be used to identify genes expressed at different levels in two tissues. Small amounts of tissue derived from two plants, one inf...

journal_title：BioTechniques

authors： Bertiol DJ,Smoker M,Brown AC,Jones MG,Burrows PR

更新日期：1994-06-01 00:00:00

abstract：:The limiting detection signal for identification of human genetic markers, such as HLA-D and VNTR genes, was determined using DNA isolated from a series of decreasing numbers of lymphocytes carrying the target marker in the polymerase chain reaction (PCR). The PCR procedure was assembled by incorporating 32P-labeled d...

journal_title：BioTechniques

authors： McDaniel DO,Naftilan J,Barber WH

更新日期：1993-07-01 00:00:00

abstract：:Under-agarose chemotaxis has been used previously to assess the ability of neutrophils to respond to gradients of chemoattractant. We have adapted this assay to the chemotactic movement of Dictyostelium amoebae in response to folic acid. Troughs are used instead of wells to increase the area along which the cells can ...

journal_title：BioTechniques

authors： Laevsky G,Knecht DA

更新日期：2001-11-01 00:00:00

abstract：:For a feasible and cost-effective impedance measurement of cellular alterations in real-time, we combined commercially available microelectrode arrays (MEAs), consisting of 60 microelectrodes, with a conventional impedance analyzer. For proof of principle, a breast carcinoma cell line (MCF-7) was cultured on MEAs, and...

journal_title：BioTechniques

authors： Rothermel A,Nieber M,Müller J,Wolf P,Schmidt M,Robitzki AA

更新日期：2006-10-01 00:00:00

abstract：:Direct contact-based coculture of human dermal fibroblasts and epidermal keratinocytes has been a long-standing and challenging issue owing to different serum and growth factor requirements of the two cell types. Existing protocols employ high serum concentrations (up to 10% fetal bovine serum), complex feeder systems...

journal_title：BioTechniques

authors： Kadam S,Vandana M,Kaushik KS

更新日期：2020-11-01 00:00:00

abstract：:Recent computational and bioinformatics advances have enabled the efficient creation of novel biocatalysts by reducing amino acid variability at hot spot regions. To further expand the utility of this strategy, we present here a tool called Multi-site Degenerate Codon Analyzer (MDC-Analyzer) for the automated design o...

journal_title：BioTechniques

authors： Tang L,Wang X,Ru B,Sun H,Huang J,Gao H

更新日期：2014-06-01 00:00:00

abstract：:Using a simple electronic circuit, a thermocouple can be connected to a chart recorder to measure the actual temperature inside a PCR tube. This allows accurate inspection of the thermocycle program and comparison between thermoprofiles from different thermocyclers. We found that the recording of temperature cycling e...

journal_title：BioTechniques

authors： Stamm S,Gillo B,Brosius J

更新日期：1991-04-01 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

authors： Webb SA

更新日期：2014-08-01 00:00:00

abstract：:We have developed a high-throughput, multiplex reverse transcription PCR (RTPCR) assay that is suitable for the analysis of medium-to low-copy cellular RNA transcripts from small numbers of cells (10(4)). High throughput was attained by utilizing microplate-based RNA extraction and RTPCR protocols, followed by PCR pro...

journal_title：BioTechniques

authors： Su S,Vivier RG,Dickson MC,Thomas N,Kendrick MK,Williamson NM,Anson JG,Houston JG,Craig FF

更新日期：1997-06-01 00:00:00

abstract：:Oligonucleotide primers used to amplify target DNA regions via PCR should meet certain design criteria to maximize the potential for efficient priming. The Random Oligonucleotide Construction Kit (ROCK), a spreadsheet-based program that runs under Microsoft Excel 97 or later version for Microsoft Windows, was develope...

journal_title：BioTechniques

authors： Strain SR,Chmielewski JG

更新日期：2001-06-01 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

authors： Said S,Tamura T,Gerdes AM

更新日期：1998-09-01 00:00:00

abstract：:We describe a convenient PCR-based protocol for in vitro recombination of homologous genes, thereby minimizing the rate of associated point mutations. High-fidelity recombination conditions were obtained using Vent DNA polymerase, which, in contrast to Taq DNA polymerase, shows significant proofreading activity and ra...

journal_title：BioTechniques

authors： Ninkovic M,Dietrich R,Aral G,Schwienhorst A

更新日期：2001-03-01 00:00:00

abstract：:The oligonucleotide ligation assay (OLA) was adapted to the genotyping of the N-arylamine-acetyltransferase (NAT2) gene. This assay allows the use of 96-well microplates and robotic workstations for high sample throughput. We found this assay to be accurate, efficient and reliable. Another advantage for epidemiologica...

journal_title：BioTechniques

authors： Bigler J,Chen C,Potter JD

更新日期：1997-04-01 00:00:00

abstract：:Quantitative PCR assays are now the standard method for viral diagnostics. These assays must be specific, as well as sensitive, to detect the potentially low starting copy number of viral genomic material. We describe a new technique, polymerase chain displacement reaction (PCDR), which uses multiple nested primers in...

journal_title：BioTechniques

authors： Harris CL,Sanchez-Vargas IJ,Olson KE,Alphey L,Fu G

更新日期：2013-02-01 00:00:00

abstract：:The use of reverse transcription (RT) PCR for relative quantitation of gene transcripts relies on the reproducibility of the individual RT, PCR and product measurement steps. Semi-competitive RT-PCR (RT-cPCR) uses an internal competitor template in the PCR step to improve quantitation. We have surveyed the reproducibi...

journal_title：BioTechniques

authors： Hall LL,Bicknell GR,Primrose L,Pringle JH,Shaw JA,Furness PN

更新日期：1998-04-01 00:00:00

abstract：:Display of functional antibody fragments on the surface of filamentous bacteriophages allows fast selection of specific phage antibodies against a variety of target antigens. However, enrichment of single chain variable fragment (scFv)-displaying phages is often hampered by the abundance of bacteriophages lacking anti...

journal_title：BioTechniques

authors： Tur MK,Huhn M,Sasse S,Engert A,Barth S

更新日期：2001-02-01 00:00:00

abstract：:Real-time or quantitative loop-mediated isothermal amplification (qLAMP) is a promising technique for the accurate detection of pathogens in organisms and the environment. Here we present a comparative study of the performance of six fluorescent intercalating dyes-SYTO-9, SYTO-13, SYTO-82, SYBR Green I, SYBR Gold, Eva...

journal_title：BioTechniques

authors： Oscorbin IP,Belousova EA,Zakabunin AI,Boyarskikh UA,Filipenko ML

更新日期：2016-07-01 00:00:00

abstract：:PhDs, postdocs, fellows and newly appointed lecturers all fall into the category of early-career researchers (ECRs). The life of researchers in early stages of their career is far from straightforward. Funding is a huge barrier for many ECRs looking to make the next step in their career, but it's not the only challeng...

journal_title：BioTechniques

authors：

更新日期：2019-01-01 00:00:00

abstract：:This report describes a common method of obtaining template DNA from phagemids, phages and plasmids. The strategy is based on the use of the cationic detergent cetyl-trimethylammonium bromide (CTAB) for DNA precipitation. By avoiding phase separation, many manipulation steps are reduced. A time-saving modification to ...

journal_title：BioTechniques

authors： Del Sal G,Manfioletti G,Schneider C

更新日期：1989-05-01 00:00:00

abstract：:The Drosophila heart has gained considerable traction as a model of cardiac development and physiology. Previously we described a semiautomatic optical heartbeat analysis (SOHA) method for quantifying functional parameters from the fly heart that facilitated its use as an organ system and disease model. Here we presen...

journal_title：BioTechniques

authors： Cammarato A,Ocorr S,Ocorr K

更新日期：2015-02-01 00:00:00

abstract：:A method is described for preparing mutants with multiple, site-directed mutations by ordered coupling of PCR-generated fragments catalyzed by a thermostable DNA ligase. Annealing of the sense strands of the fragments to a single-stranded (antisense) template created a full-length sense strand leaving only nicks that ...

journal_title：BioTechniques

authors： Rouwendal GJ,Wolbert EJ,Zwiers LH,Springer J

更新日期：1993-07-01 00:00:00