Adaptation of the diphenylamine (DPA) assay to a 96-well plate tissue culture format and comparison with the MTT assay.

abstract：:Directed evolution is poised to change small molecule discovery and provide greater access to "drug space." Sarah Webb looks into the evolving drug discovery landscape. ...

journal_title：BioTechniques

authors： Webb S Ph D

更新日期：2017-12-01 00:00:00

abstract：:Quantitative real-time PCR has become a popular method to analyze and quantify changes in the copy number of mitochondrial DNA (mtDNA), and nuclear DNA (nDNA) is often used as an endogenous reference for mtDNA abundance. In our experience, using nDNA as a reference is problematic, due to differences in the extraction ...

journal_title：BioTechniques

authors： Myers MB,Mittelstaedt RA,Heflich RH

更新日期：2009-10-01 00:00:00

abstract：:Recent advances in long reverse transcription (RT)-PCR technology allow the copying of full-length coding regions of large mRNAs in one step. Using long RT-PCR, one can be certain that a given cDNA is derived from a single mRNA. In what to our knowledge is a novel application, we can isolate and characterize splice va...

journal_title：BioTechniques

authors： Hu Y,Tanzer LR,Cao J,Geringer CD,Moore RE

更新日期：1998-08-01 00:00:00

abstract：:Current protocols for screening proliferative factors for β cells ex vivo are time-consuming, require cell lines or dissociated islets, and often entail expensive specialized screening equipment. Here we present an efficient and lower cost alternative that utilizes intact mouse islets for the initial screening of prol...

journal_title：BioTechniques

authors： Mosser RE,Gannon M

更新日期：2013-12-01 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

authors： Amaravadi L,King MW

更新日期：1994-01-01 00:00:00

abstract：:As a strategy to purify recombinant murine Interleukin (IL)-18, we cloned the mature coding region of this protein into the pFLAG-1 expression system. The intent was to use the FLAG peptide "tag" as an amino terminal addition to IL-18 so that purification of this fusion protein (FLAG-IL-18) on anti-FLAG antibody affin...

journal_title：BioTechniques

authors： Elhofy A,Bost KL

更新日期：1998-09-01 00:00:00

abstract：:Bovine serum albumin, free of deoxyribonuclease activity, was obtained in our laboratory using ion-exchange chromatography followed by acetylation. Chromatography on four different resins (DEAE-52, P-11, hydroxylapatite and Q Sepharose fast-flow) was examined. Fractions from Q Sepharose chromatography, eluted with a l...

journal_title：BioTechniques

authors： Trujillo LE,Castellanos L,Garcia O,Herrera L

更新日期：1990-11-01 00:00:00

abstract：:We describe a novel assay format for the Gal4-based yeast two-hybrid-system, in which the readout from three different reporter genes is measured sequentially in a single microplate. Activation of the URA3, MEL1, and lacZ reporters in response to a protein-protein interaction is monitored by measuring sequentially: (i...

journal_title：BioTechniques

authors： Evans DR,Swirsding KA,Taillon BE,Simons JF

更新日期：2004-11-01 00:00:00

abstract：:We describe a bead-based, multiplexed, oligonucleotide ligation assay (OLA) performed on the Luminex flow cytometer. Differences between this method and those previously reported include the use of far fewer beads and the use of a universal oligonucleotide for signal detection. These innovations serve to significantly...

journal_title：BioTechniques

authors： Bruse S,Moreau M,Azaro M,Zimmerman R,Brzustowicz L

更新日期：2008-11-01 00:00:00

abstract：:As an improved strategy for producing functional macromolecules by in vitro evolutionary optimization, we propose an automated machine that can process up to 960 samples in parallel. It consists of a 960-well PCR machine with special sealed plastic reaction vessels and appropriate handling devices. We show that the he...

journal_title：BioTechniques

authors： Schober A,Walter NG,Tangen U,Strunk G,Ederhof T,Dapprich J,Eigen M

更新日期：1995-04-01 00:00:00

abstract：:The type IIS/IIC restriction endonuclease TspGWI recognizes the sequence 5'-ACGGA-3', cleaving DNA 11/9 nucleotides downstream. Here we show that sinefungin, a cofactor analog of S-adenosyl methionine, induces a unique type of relaxation in DNA recognition specificity. In the presence of sinefungin, TspGWI recognizes ...

journal_title：BioTechniques

authors： Zylicz-Stachula A,Zołnierkiewicz O,Jeżewska-Frąckowiak J,Skowron PM

更新日期：2011-06-01 00:00:00

abstract：:To move microarray technology into the diagnostic realm, the impact of technical parameters, such as sample preparation and RNA extraction, needs to be understood and minimized. We evaluated the impact of two RNA extraction methods, DNase treatment and the amount of hybridized cDNA probe, on the outcome of microarray ...

journal_title：BioTechniques

authors： Ojaniemi H,Evengard B,Lee DR,Unger ER,Vernon SD

更新日期：2003-11-01 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：:Pairs of primers flanking known miniTn10 transposon insertion sites were used to confirm the presence of the transposon in DNA isolated from Legionella pneumophila mutants. It was expected that the polymerase chain reaction products derived from the mutant template would be larger than those from the wild-type (WT) te...

journal_title：BioTechniques

authors： Viswanathan VK,Krcmarik K,Cianciotto NP

更新日期：1999-09-01 00:00:00

abstract：:There is an enormous initiative to establish the genetic basis for disorders of brain function. Unfortunately, genetic intervention is not accomplished easily in the nervous system. One strategy is to engineer and deliver to neurons specialized viral vectors that carry a gene (or genes) of interest, thereby exploiting...

journal_title：BioTechniques

authors： Neve RL,Neve KA,Nestler EJ,Carlezon WA Jr

更新日期：2005-09-01 00:00:00

abstract：:Screening methods for chemotherapeutic agents usually rely on the cytotoxic properties of the drugs. However, agents that inhibit invasion may have more efficacy and cause fewer side effects. Various cellular invasion assays have been used to evaluate these types of compounds, including the modified Boyden chamber, mo...

journal_title：BioTechniques

authors： Sasaki CY,Passaniti A

更新日期：1998-06-01 00:00:00

abstract：:We describe the application of simple cloning by prolonged overlap extension for multiple site-directed mutagenesis in the same plasmid. We show that it is possible to use this technique with very short PCR templates. The technique is ideally suited for the generation of longer donor DNA sequences for CRISPR/Cas9-medi...

journal_title：BioTechniques

authors： Hejlesen R,Füchtbauer EM

更新日期：2020-06-01 00:00:00

abstract：:Methods have been developed to rapidly visualize the size distribution of third complementarity-determining regions (CDR3) in immunoglobulin (Ig) and T-cell receptor (TCR) molecules. DNA fragments spanning the Ig or TCR CDR3 are generated by PCR using primers at fixed positions in the variable and constant segments. T...

journal_title：BioTechniques

authors： Raaphorst FM,Tami J,Sanz IE

更新日期：1996-01-01 00:00:00

abstract：:Recombinant alphaviruses have been used as vehicles for delivery and expression of heterologous genes in mammalian, avian and insect cell lines. We have used a Sindbis replicon virus (Sinreplac) able to express the E. coli lacZ gene to compare the efficiency of transduction in one insect, six mammalian cell lines and ...

journal_title：BioTechniques

authors： Corsini J,Traul DL,Wilcox CL,Gaines P,Carlson JO

更新日期：1996-09-01 00:00:00

abstract：:We report a straightforward methodology for purification of recombinant proteins by incorporating a short hydrophilic peptide marker segment at their N-termini. A calcium-dependent antibody that reacts primarily with the first three amino acids of this peptide segment was used to affinity purify the fusion proteins in...

journal_title：BioTechniques

authors： Prickett KS,Amberg DC,Hopp TP

更新日期：1989-06-01 00:00:00

abstract：:We report a robust method for studying en masse changes in translation using cDNA arrays. The relative distribution of messenger RNAs (mRNAs) along polysome gradients was monitored by performing cDNA array analysis of each gradient fraction and quantifying the mRNA translational status by regression analysis. Using th...

journal_title：BioTechniques

authors： Mazan-Mamczarz K,Kawai T,Martindale JL,Gorospe M

更新日期：2005-07-01 00:00:00

abstract：:Inverse PCR has been used for the recovery of genome regions flanking a known sequence, although its application to metagenome walking is limited due to inefficient amplification from low copy number fragments. Here we present an improved inverse PCR scheme that enables walking of rare fragments in environmental metag...

journal_title：BioTechniques

authors： Uchiyama T,Watanabe K

更新日期：2006-08-01 00:00:00

abstract：:Sample automation and management is increasingly important as the number and size of population-scale and high-throughput projects grow. This is particularly the case in large-scale population studies where sample size is far outpacing the commonly used 96-well plate format. To facilitate management and transfer of sa...

journal_title：BioTechniques

authors： Cario CL,Witte JS

更新日期：2018-12-01 00:00:00

abstract：:Manipulating gene expression in primary neurons has been a goal for many scientists for over 20 years. Vertebrate central nervous system neurons are classically difficult to transfect. Most lipid reagents are inefficient and toxic to the cells, and time-consuming methods such as viral infections are often required to ...

journal_title：BioTechniques

authors： Buchser WJ,Pardinas JR,Shi Y,Bixby JL,Lemmon VP

更新日期：2006-11-01 00:00:00

abstract：:Reverse transcription PCR (RT-PCR) represents a sensitive and powerful tool for analyzing RNA. While it has tremendous potential for quantitative applications, a comprehensive knowledge of its technical aspects is required. Successful quantitative RT-PCR involves correction for experimental variations in individual RT...

journal_title：BioTechniques

authors： Freeman WM,Walker SJ,Vrana KE

更新日期：1999-01-01 00:00:00

abstract：:Fluorescent live imaging of cells and embryos at subcellular resolution poses significant challenges for biologists due to morbidity and mortality ensuing from phototoxicity. Here we report the use of a spinning-disk confocal microscope to image mouse and bovine preimplantation embryos without impairing their developm...

journal_title：BioTechniques

authors： Ross PJ,Perez GI,Ko T,Yoo MS,Cibelli JB

更新日期：2006-12-01 00:00:00

abstract：:Here we report a simple new method for exposing cells to normoxic and hypoxic conditions using vacuum bags, normally employed for food storage, to establish and maintain low oxygen levels in vitro. Vacuum bags were gassed with a mixture containing specified levels of oxygen, then sealed, creating a hypoxic microenviro...

journal_title：BioTechniques

authors： Bakmiwewa SM,Heng B,Guillemin GJ,Ball HJ,Hunt NH

更新日期：2015-10-01 00:00:00

abstract：:A new genetic marker was created in which sequences from enhanced green fluorescent protein were fused to those of puromycin N-acetyl transferase. The resulting fusion protein (EGFP-puro) conferred both green fluorescence and resistance to puromycin when expressed in mammalian cells. The utility of EGFP-puro as a sele...

journal_title：BioTechniques

authors： Abbate J,Lacayo JC,Prichard M,Pari G,McVoy MA

更新日期：2001-08-01 00:00:00

abstract：:Comprehensive analysis of DNA methylation patterns is critical for understanding the molecular basis of many human diseases. While hundreds of PCR-based DNA methylation studies are published every year, the selection and implementation of appropriate methods for these studies can be challenging for molecular genetics ...

journal_title：BioTechniques

authors： Hernández HG,Tse MY,Pang SC,Arboleda H,Forero DA

更新日期：2013-10-01 00:00:00

abstract：:We present a fast, convenient and inexpensive method that allows the automated, large-scale screening of chemical libraries for compounds that are converted by the herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) into inhibitors of cell growth. The method is based on the use of budding yeast (Saccharomyces ce...

journal_title：BioTechniques

authors： Wera S,Degrève B,Balzarini J,De Clercq E,Thevelein JM,Neyts J

更新日期：1999-10-01 00:00:00