Two large insert vectors, lambda PS and lambda KO, facilitate rapid mapping and targeted disruption of mammalian genes.

abstract：:A modification of PCR-mediated gene synthesis strategy is introduced. This modification enables the synthesis of a gene from oligonucleotides comprising only one of the two strands. Bridging oligonucleotides (approximately 20-mers in length) complementary to the junctions of template strand oligonucleotides and two ou...

journal_title：BioTechniques

authors： Jayaraman K,Puccini CJ

更新日期：1992-03-01 00:00:00

abstract：:The green fluorescent protein (GFP) from the jellyfish Aequorea victoria is rapidly becoming an important reporter molecule for monitoring gene expression and protein localization in vivo, in situ and in real time. GFP emits bright green light (lambda max = 509 nm) when excited with UV or blue light (lambda max = 395 ...

journal_title：BioTechniques

authors： Kain SR,Adams M,Kondepudi A,Yang TT,Ward WW,Kitts P

更新日期：1995-10-01 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

authors： Copley CG,Boot C

更新日期：1992-12-01 00:00:00

abstract：:We have altered the antibiotic resistance of the reporter plasmids and the pJG4-5 activation-domain and pEG202 DNA binding-domain plasmids used in the Brent interaction trap/two-hybrid system. These plasmids were each previously ampicillin-resistant, resulting in an inefficient purification of any one plasmid from a y...

journal_title：BioTechniques

authors： Watson MA,Buckholz R,Weiner MP

更新日期：1996-08-01 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：:Vascular targeting agents (VTAs) can be produced by linking antibodies or antibody fragments directed against endothelial cell markers to effector moieties. So far, it has been necessary to produce the components of VTAs (antibody, antibody fragment, linker, and effector) separately and, subsequently, to conjugate the...

journal_title：BioTechniques

authors： Gottstein C,Wels W,Ober B,Thorpe PE

更新日期：2001-01-01 00:00:00

abstract：:Array technology is a widely used tool for gene expression profiling in various biological systems. However, the application of this method to mammalian preimplantation embryos is limited by the small amount of mRNA that can be extracted from a single embryo, which is not sufficient for array analysis. Here we report ...

journal_title：BioTechniques

authors： Brambrink T,Wabnitz P,Halter R,Klocke R,Carnwath J,Kues W,Wrenzycki C,Paul D,Niemann H

更新日期：2002-08-01 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

authors： Uhlén M

更新日期：2008-04-01 00:00:00

abstract：:Genetic immunization is an attractive approach to generate antibodies because native proteins are expressed in vivo with normal posttranscriptional modifications, avoiding time-consuming and costly antigen isolation or synthesis. Hydrodynamic tail or limb vein delivery of naked plasmid DNA expression vectors was used ...

journal_title：BioTechniques

authors： Bates MK,Zhang G,Sebestyén MG,Neal ZC,Wolff JA,Herweijer H

更新日期：2006-02-01 00:00:00

abstract：:Hematopoietic stem cells (HSCs) express Mdr1a/1b and Bcrp1/Abcg2, which are members of the ATP binding cassette transporter family. Mice lacking both Mdr1-type genes (Mdr1a and Mdr1b) or Bcrp1 had normal hematopoietic development, but it has been unclear whether Mdr1a/1b and Bcrp1 play redundant roles in hematopoiesis...

journal_title：BioTechniques

authors： Zhou S,Zong Y,Lu T,Sorrentino BP

更新日期：2003-12-01 00:00:00

abstract：:We have developed a rapid, sensitive, and quantitative 96-well microplate-based nonradioactive immunoprecipitation/kinase assay to evaluate mitogen-activated protein kinase (MAPK) activity. Three quantitative nonradioactive imunoprecipitation/kinase assays of MAPK were demonstrated on a 96-well microplate coated with ...

journal_title：BioTechniques

authors： Zhang L,Uder S,Juehne T,Brizzard B,Song K

更新日期：2002-02-01 00:00:00

abstract：:Automated DNA sequencing requires the intensive use of computers to handle the large amount of data taken. When a computer failure occurs and the data are no longer accessible, all the expense and effort that went into the sequencing experiment is lost. By using the data storage architecture of Macintosh computers to ...

journal_title：BioTechniques

authors： O'Brien KM,Fondon JW 3rd,Evans GA,Garner HR

更新日期：1997-06-01 00:00:00

abstract：:Perfluorocarbon emulsions were applied to hybridoma cultures grown in tissue culture tubes and column bioreactors. The oxygen transfer enhancement effect of perfluorocarbon emulsions was clearly demonstrated by the higher cell densities obtained in emulsion-supplemented systems. In addition, perfluorocarbon emulsions ...

journal_title：BioTechniques

authors： Ju LK,Armiger WB

更新日期：1992-02-01 00:00:00

abstract：:We previously reported that cervicovaginal lavages (CVL) contain a factor that enhances the replication of human immunodeficiency virus (HIV) by increasing virus transcription in T cells and monocytoid cells. This factor was named the HIV-inducing factor (HIF). To determine the molecular mass of HIF, we adapted a blot...

journal_title：BioTechniques

authors： Hashemi FB,Spear GT,Madsen L,Mollenhauer J

更新日期：2000-03-01 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

authors： Ohara R,Kikuno RF,Kitamura H,Ohara O

更新日期：2005-03-01 00:00:00

abstract：:We have developed a novel method for the rapid preparation of large quantities of 3' end-labeled single-stranded (ss) DNA (ssDNA) probes for transcript mapping. A recombinant phagemid vector containing the probe sequence was used to raise large quantities of ssDNA. Based on the DNA sequence of the probe, an oligonucle...

journal_title：BioTechniques

authors： Silk GW,Wu M

更新日期：1993-05-01 00:00:00

abstract：:A method was developed for the detection of bacterial mRNAs using reverse transcriptase followed by the polymerase chain reaction (PCR) and Southern blot analysis. The method involves brief inhibition of protein synthesis with chloramphenicol, followed by reverse transcription, PCR amplification of cDNA and Southern b...

journal_title：BioTechniques

authors： Mahbubani MH,Bej AK,Miller RD,Atlas RM,DiCesare JL,Haff LA

更新日期：1991-01-01 00:00:00

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

authors： Nicolaides NC,Stoeckert CJ Jr

更新日期：1990-02-01 00:00:00

abstract：:Subtractive hybridization has been widely used for the identification of differentially expressed genes. Here we describe a simple, sensitive strategy of subtractive hybridization that involves binding the driver poly(A)+ RNA pool to paramagnetic Dynabeads Oligo (dT)25. After hybridization with target cDNA, the molecu...

journal_title：BioTechniques

authors： Mészáros M,Morton DB

更新日期：1996-03-01 00:00:00

abstract：:Identification of antimitotic molecules that affect tubulin dynamics is a multistep procedure. It includes in vitro tubulin polymerization assay, studies of a cell cycle effect, and general cytotoxicity assessment. To simplify this lengthy screening protocol, we have introduced and validated an assay system based on t...

journal_title：BioTechniques

authors： Semenova MN,Kiselyov A,Semenov VV

更新日期：2006-06-01 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

authors： Ilagan R,Guthrie K,Quinlan S,Rapoport HS,Jones S,Church A,Basu J,Ludlow J

更新日期：2010-02-01 00:00:00

abstract：:A fully automated nucleic acid analysis system is described, which offers positive sample identification, improved sensitivity and reduced user interaction compared to conventional techniques. The system relies on the sequence-specific capture of DNA onto solid-phase particles, confirming product identity without the ...

journal_title：BioTechniques

authors： Brown A,Akinsanya AA,Barker SJ,Brophy M,Dobb AK,Doyle SM,Hudson IR,Minter SJ,Wraith MJ,Oultram JD

更新日期：1999-07-01 00:00:00

abstract：:GC-rich DNA regions were PCR-amplified with Taq DNA polymerase using either the canonical set of deoxynucleoside triphosphates or mixtures in which the dCTP had been partially or completely replaced by its N4-methylated analog, N4-methyl-2'-deoxycytidine 5'-triphosphate (N4me-dCTP). In the case of a particularly GC-ri...

journal_title：BioTechniques

authors： Flores-Juárez CR,González-Jasso E,Antaramian A,Pless RC

更新日期：2016-10-01 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：: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：: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：:Microarray expression analysis has become one of the most widely used functional genomics tools. Efficient application of this technique requires the development of robust and reproducible protocols. We have optimized all aspects of the process, including PCR amplification of target cDNA clones, microarray printing, p...

journal_title：BioTechniques

authors： Hegde P,Qi R,Abernathy K,Gay C,Dharap S,Gaspard R,Hughes JE,Snesrud E,Lee N,Quackenbush J

更新日期：2000-09-01 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：:Monitoring morphogenetic processes, at high resolution over time, has been a long-standing goal of many developmental cell biologists. It is critical to image cells in their natural environment whenever possible; however, imaging many warm-blooded vertebrates, especially mammals, is problematic. At early stages of dev...

journal_title：BioTechniques

authors： Rupp PA,Rongish BJ,Czirok A,Little CD

更新日期：2003-02-01 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

authors： Taatjes DJ,Palmer CJ,Pantano C,Hoffmann SB,Cummins A,Mossman BT

更新日期：2001-10-01 00:00:00