Screening recombinant DNA libraries: a rapid and efficient method for isolating cDNA clones utilizing the PCR.

abstract：:Genome sequencing projects are either based on whole genome shotgun (WGS) or on a BAC-by-BAC strategy. Although WGS is in most cases the preferred choice, sometimes the BAC-by-BAC approach may be better because it requires a much simpler assembly process. Furthermore, when the study is limited to specific regions of t...

journal_title：BioTechniques

authors： Todesco S,Campagna D,Levorin F,D'Angelo M,Schiavon R,Valle G,Vezzi A

更新日期：2008-01-01 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：:DNA sequences containing tandem dinucleotide repeats represent an abundant source of DNA polymorphism in human and other eukaryotic genomes. Here we describe a novel technique for the identification and characterization of regions of DNA containing these repetitive elements. Using primers designed to recognize tandem ...

journal_title：BioTechniques

authors： Grist SA,Firgaira FA,Morley AA

更新日期：1993-08-01 00:00:00

abstract：:One of the challenges of performing live-cell imaging in plants is establishing a system for securing the sample during imaging that allows for the rapid addition of treatments. Here we report how a commercially available device called a HybriWell™ can be repurposed to create an imaging chamber suitable for Arabidopsi...

journal_title：BioTechniques

authors： Vang S,Seitz K,Krysan PJ

更新日期：2018-06-01 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

authors： Novák P,Gaburjáková M,Zahradník I

更新日期：2007-03-01 00:00:00

abstract：:The use of quantitative PCR is recommended to monitor the level of residual hematological malignancies. The proposed multiplex IgH/ras PCR uses a co-amplification of the clonal CDR3 rearrangement of the immunoglobulin heavy chain gene (IgH) as a disease marker and a segment of the Hras 1 gene containing codon 61 (ras)...

journal_title：BioTechniques

authors： Slavícková A,Ullmannová V,Klener P

更新日期：2000-04-01 00:00:00

abstract：:The technique described here is a fast and simple method of extracting chloroplast DNA (cpDNA). It overcomes the need for differential centrifugation using density gradients. The leaves do not have to be kept in the dark and lyophilized before extraction, but lyophilization is still possible. The chloroplasts are spec...

journal_title：BioTechniques

authors： Mariac C,Trouslot P,Poteaux C,Bezançon G,Renno JF

更新日期：2000-01-01 00:00:00

abstract：:Here we report the construction of a histidine-tagged T4 RNA ligase expression plasmid (pRHT4). The construct, when overexpressed in BL21 (DE3) cells, allows the preparation of large quantities of T4 RNA ligase in high purity using only a single purification column. The histidine affinity tag does not inhibit enzyme f...

journal_title：BioTechniques

authors： Wang QS,Unrau PJ

更新日期：2002-12-01 00:00:00

abstract：:A pipeline has been created for the characterization of Arabidopsis thaliana mutants by generating flanking sequence tags (FSTs) and optimized for economic, high-throughput production. The GABI-Kat collection of T-DNA mutagenized A. thaliana plants was used as a source of independent transgenic lines. The pipeline inc...

journal_title：BioTechniques

authors： Strizhov N,Li Y,Rosso MG,Viehoever P,Dekker KA,Weisshaar B

更新日期：2003-12-01 00:00:00

abstract：:We describe a method for rapid radioactive labeling of PCR product. The method, employing the Klenow fragment of DNA polymerase I, consumes little product, requires no product purification and takes under 30 minutes. ...

journal_title：BioTechniques

authors： McDaniel TK,Huang Y,Yin J,Needleman SW,Meltzer SJ

更新日期：1991-08-01 00:00:00

abstract：:Recombinant retroviruses are a common vehicle to deliver an exogenous gene to a target cell, which makes them a useful tool in the field of gene therapy. A major drawback to using recombinant retroviruses is the low titer achieved, resulting in a limited number of target cells infected and subsequently poor expression...

journal_title：BioTechniques

authors： Tobias CA,Kim D,Fischer I

更新日期：2000-10-01 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

authors： Yang YS,Hughes TE

更新日期：2001-11-01 00:00:00

abstract：:Antibiotics are widely useful in medicine, agriculture, and industrial fermentations. However, increasing problems with resistant strains call for restrained use and alternative strategies. Antisense peptide nucleic acids (PNAs) show potent bactericidal effects when targeted against the essential Escherichia coli acpP...

journal_title：BioTechniques

authors： Dryselius R,Nekhotiaeva N,Nielsen PE,Good L

更新日期：2003-11-01 00:00:00

abstract：:Site-specific mutagenesis and directional subcloning were accomplished by using the polymerase chain reaction to generate products that can recombine to form circular DNA. This DNA was transfected into E. coli without phosphorylation of primers, restriction enzyme digestion or ligation. Specifically, the polymerase ch...

journal_title：BioTechniques

authors： Jones DH,Howard BH

更新日期：1990-02-01 00:00:00

abstract：:As more and more researchers are examining proteins that are available only in extremely limited quantities, i.e., cellular extracts or genetic engineering products, it is critical to utilize staining methods that maximize sensitivity. The protocol we describe here--double staining of polyacrylamide electrophoresis ge...

journal_title：BioTechniques

authors： Ross M,Peters L

更新日期：1990-11-01 00:00:00

abstract：:The CRISPR/Cas9 system is an efficient gene-editing method, but it is difficult to obtain mutants for some specific species and special genome structures. A previously reported multiplexed, semi-nested PCR target-enrichment approach, which does not rely on transgenic technology, has been shown to be an effective and a...

journal_title：BioTechniques

authors： Zhang Y,Chi X,Feng L,Wu X,Qi X

更新日期：2019-12-01 00:00:00

abstract：:Autoantibodies directed against the 68-kDa (U1) ribonucleoprotein antigen are mainly found in sera of patients with mixed connective tissue disease. The corresponding cDNA was fragmented into four regions coding for the major antigenic epitopes A', B', C' and D'. All the epitopes were subcloned and expressed as fusion...

journal_title：BioTechniques

authors： Frorath B,Scanarini M,Netter HJ,Abney CC,Liedvogel B,Lakomek HJ,Northemann W

更新日期：1991-09-01 00:00:00

abstract：:Sequencing of the human genome in combination with computational annotation has provided tremendous data on predicted genes. However, for most of them, no corresponding cDNAs are available yet. Furthermore, even where cDNA clones were obtained, gene transcripts often have many different splice variants that are not co...

journal_title：BioTechniques

authors： Mitani Y,Nakayama T,Harbers M,Hayashizaki Y

更新日期：2004-07-01 00:00:00

abstract：:The two-hybrid system in Saccharomyces cerevisiae is a genetic approach for the detection of of protein-protein interactions in vivo. This technology relies on the the activity of separated DNA-binding and transactivation domains of specific transcription factors to reconstitute an active transcription factor complex ...

journal_title：BioTechniques

authors： Mayer G,Launhardt H,Munder T

更新日期：1999-07-01 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：:Multiplex Manager 1.0 is a user-friendly cross-platform program that designs efficient combinations of existing genetic marker loci into multiplex polymerase chain reactions and optimizes using prior marker information. The program has the flexibility to solve two design problems: combining all markers into the smalle...

journal_title：BioTechniques

authors： Holleley CE,Geerts PG

更新日期：2009-06-01 00:00:00

abstract：:Methylation of DNA in CpG dense regions of gene promoters (CpG islands) is important for transcriptional inactivation of selective genes in normal and neoplastic cells. Here, we present a spreadsheet-based program adapted from Microsoft Excel that is useful for identifying CpG islands and for assisting in the laborato...

journal_title：BioTechniques

authors： Anbazhagan R,Herman JG,Enika K,Gabrielson E

更新日期：2001-01-01 00:00:00

abstract：:The use of AlphaScreen® detection has allowed researchers to examine a wide variety of molecular interactions for use in high-throughput screening. However, the cost of Alpha reagents can often be prohibitory for extended screening campaigns or for young investigators with limited funding. To reduce assay costs, many ...

journal_title：BioTechniques

authors： Veloria JR,Devkota AK,Cho EJ,Dalby KN

更新日期：2018-04-01 00:00:00

abstract：:The ZEBRA protein is a transcriptional activator that induces expression of viral lytic genes in cells harboring latent Epstein-Barr virus (EBV). In this report it is shown that a derivative of ZEBRA that cannot activate transcription (Zd) can be used to detect and characterize activation domains. Three expression vec...

journal_title：BioTechniques

authors： Asković S,Baumann R

更新日期：1997-05-01 00:00:00

abstract：:The degree of fluorescence polarization (FP) of a fluorescent molecule is a reflection of its molecular weight (Mr). FP is therefore a useful detection methodfor homogeneous assays in which the starting reagents and products differ significantly in Mr. We have previously shown that FP is a good detection method for th...

journal_title：BioTechniques

authors： Hsu TM,Chen X,Duan S,Miller RD,Kwok PY

更新日期：2001-09-01 00:00:00

abstract：:The PCR method has proved to be an invaluable tool for the specific and sensitive detection of genetically modified material (e.g., Roundup Ready Soybean and Bt-176 "Maximizer" Maize) in foodstuffs. The first step in the procedure, namely the purification of nucleic acids from the sample, is often the deciding factor ...

journal_title：BioTechniques

authors： Tengel C,Schüssler P,Setzke E,Balles J,Sprenger-Haussels M

更新日期：2001-08-01 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：:Millions of museum specimens are integral to biodiversity studies; however, DNA degradation may limit the ability to obtain DNA sequences. In this study, a degradation analysis model for Lepidoptera specimens was established. Based on this model, we revealed the characteristics of DNA fragment distribution caused by e...

journal_title：BioTechniques

authors： Xu Y,Ren X,Wang H,Wang M,Li G

更新日期：2020-03-01 00:00:00

abstract：:Sensitive nucleic acid based detection methods such as in situ PCR, in situ RT-PCR and PRINS have great potential in the areas of developmental biology, pathogenesis and diagnostics. However, control of evaporation from in situ reactions is critical to ensure reliable data. Self-Seal Reagent, a component added directl...

journal_title：BioTechniques

authors： Sullivan DE,Bobroski LE,Bagasra O,Finney M

更新日期：1997-08-01 00:00:00

abstract：:Genotyping fish larvae is a valuable technique for numerous fields of study. While methods for collecting DNA from early stage larvae have been published, a non-lethal, non-invasive genotyping protocol for hatchlings that is amenable to high-throughput approaches is desirable. Here, we describe a method to individuall...

journal_title：BioTechniques

authors： Espinoza GJ,Poland JM,Bremer JRA

更新日期：2017-10-01 00:00:00