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 yeast strain containing all three plasmids that constitute the complete interaction trap. By creating derivatives of each of these plasmids expressing either kanamycin or chloramphenicol resistance, along with the parent plasmids, we now have the option to use the interaction trap in yeast with three E. coli differentially selectable vectors. This will allow isolation of any one plasmid by purifying all of the interaction trap plasmids from yeast simultaneously and plating E. coli transformed with the plasmids onto the appropriate antibiotic plate to select the particular plasmid of interest.
journal_name
Biotechniquesjournal_title
BioTechniquesauthors
Watson MA,Buckholz R,Weiner MPdoi
10.2144/96212st02subject
Has Abstractpub_date
1996-08-01 00:00:00pages
255-9issue
2eissn
0736-6205issn
1940-9818journal_volume
21pub_type
杂志文章相关文献
BIOTECHNIQUES文献大全abstract::Here we demonstrate the use of a mammalian two-hybrid system to study protein-protein interactions. Like the yeast two-hybrid system, this is a genetic, in vivo assay based on the reconstitution of the function of a transcriptional activator. In this system, one protein of interest is expressed as a fusion to the Gal4...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/97222pf02
更新日期:1997-02-01 00:00:00
abstract::Current gene synthesis methods often incorporate a PCR amplification step in order to yield final material sufficient for resolution from multiple off-products. These amplification steps can cause stochastic sampling effects that propagate errors in gene synthesis or decrease variability when applied to the constructi...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/000114330
更新日期:2015-09-01 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
pub_type: 杂志文章
doi:
更新日期:1991-09-01 00:00:00
abstract::We have modified the terminal deoxynucleotidyl transferase (TDT)-mediated dUTP-biotin nick end-labeling (TUNEL) method to permit the immunogold-silver intensification detection of apoptotic cells in tissue sections. Such sections can subsequently be processed for multi-labeling fluorescence microscopy, thus permitting...
journal_title:BioTechniques
pub_type: 杂志文章
doi:
更新日期:1995-11-01 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
pub_type: 杂志文章
doi:10.2144/btn-2019-0148
更新日期:2020-06-01 00:00:00
abstract::Conformation-sensitive gel electrophoresis (CSGE) has been introduced as the most reliable method for the screening of large and multi-exon genes because of its simplicity, sensitivity and specificity. Based on heteroduplex formation and with the use of mildly denaturing solvents, it allows detection of single-base mu...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/00285rr08
更新日期:2000-05-01 00:00:00
abstract::Impurity assays for recombinant protein therapeutics are essential to ensure batch-to-batch consistency and to meet the FDA's criteria for a well-characterized biopharmaceutical. For determination of residual host cell DNA, membrane hybridization assays utilizing radiolabeled DNA probes prepared from the host cell's g...
journal_title:BioTechniques
pub_type:
doi:10.2144/02325dd06
更新日期:2002-05-01 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
pub_type: 杂志文章
doi:
更新日期:1991-08-01 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
pub_type: 杂志文章
doi:10.2144/000112310
更新日期:2006-12-01 00:00:00
abstract::Previous work from this laboratory has shown that immunoporation has the potential for the selective transfection of a range of different animal cells based on their immunological identity. The unique ability of immunoporation to target cells for transfection combined with the high efficiency of transfection and the h...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/04372RR05
更新日期:2004-08-01 00:00:00
abstract::A nonradioactive functional assay was developed to quantitate DNA binding proteins. The assay was designed to allow the use of 96-well microplates for high sample throughput. We show that the assay can measure sequence-specific DNA binding of purified proteins as well as DNA binding activity present in whole cell extr...
journal_title:BioTechniques
pub_type:
doi:
更新日期:1995-06-01 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
pub_type:
doi:
更新日期:1994-06-01 00:00:00
abstract::Here we describe a method for the isolation of PCR-ready genomic DNA from various zebrafish tissues that is based on a previously published murine protocol. The DNA solutions are of sufficient quality to allow PCR detection of transgenes from all commonly used zebrafish tissues. In sperm, transgene amplification was s...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/000112619
更新日期:2007-11-01 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
pub_type: 杂志文章
doi:10.2144/000113156
更新日期:2009-06-01 00:00:00
abstract::Transfection of suspension cells has proven to be very difficult using conventional methods. Here, we present a simple and time-saving new transfection protocol wherein cell culture plates coated with chicken egg white are seeded with suspension cells prior to transfection. Our results demonstrate that coupling egg wh...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/000113914
更新日期:2012-08-01 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
pub_type: 杂志文章
doi:
更新日期:1989-05-01 00:00:00
abstract::We describe a general method for making template DNA for sequencing of PCR products. The procedure may be particularly useful for PCR products where minimal sequence information is known or as an alternative to primer walking when sequencing long PCR products. A cassette containing the hybridization site for the M13 s...
journal_title:BioTechniques
pub_type:
doi:
更新日期:1994-11-01 00:00:00
abstract::Radiolabeled proteins are used in a variety of laboratory applications as well as in radioimmunotherapy. This review focuses on methods that utilize genetic engineering to introduce exogenous phosphorylation sites into proteins. Protein kinase substrate sites can be introduced into target proteins to serve as tags for...
journal_title:BioTechniques
pub_type: 杂志文章,评审
doi:
更新日期:2002-10-01 00:00:00
abstract::Microsatellite sequences are important markers for population genetics studies. In the past, the development of adequate microsatellite primers has been cumbersome. However with the advent of next-generation sequencing technologies, marker identification in genomes of non-model species has been greatly simplified. Her...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/000114104
更新日期:2013-11-01 00:00:00
abstract::Patents play an increasingly important role in the dissemination of information in many fast moving fields such as biotechnology and semiconductors. Quite a few new developments are introduced as patents, and only later, if at all, do they find their way into the scientific literature. In spite of this, patents lack w...
journal_title:BioTechniques
pub_type: 杂志文章
doi:
更新日期:1988-02-01 00:00:00
abstract::Real-time PCR is becoming a preferred method for quantification of minute amounts of nucleic acids. To achieve the full potential of this technique, accurate and convenient models for post-PCR data analysis are required. In this study, three different models were chosen to quantify the definitive copy numbers of Cucum...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/000112750
更新日期:2008-06-01 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
pub_type: 杂志文章
doi:10.2144/01302rr04
更新日期:2001-02-01 00:00:00
abstract::Protein analysis is crucial to elucidating the function of proteins and understanding the impact of their presence, absence and alteration. This is key to advancing knowledge about diseases, providing the opportunity for biomarker discovery and development of therapeutics. In this issue of Tech News, Nawsheen Boodhun ...
journal_title:BioTechniques
pub_type: 新闻
doi:10.2144/btn-2018-0055
更新日期:2018-05-01 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::Address correspondence to Martin A. Kennedy, Department of Pathology & Carney Centre for Pharmacogenomics, University of Otago, Christchurch, PO Box 4345, Christchurch, New Zealand. E-mail: martin.kennedy@otago.ac.nz. ...
journal_title:BioTechniques
pub_type: 信件
doi:10.2144/000114481
更新日期:2016-12-01 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
pub_type: 杂志文章
doi:10.2144/000114255
更新日期:2015-02-01 00:00:00
abstract::Silencing of mammalian gene expression by RNA interference (RNAi) technology can be achieved using small interfering RNA (siRNA) or short hairpin RNA (shRNA). However, the relative effectiveness of these two approaches is not known. It is also not clear whether gene-specific shRNA transcribed from an RNA polymerase II...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/04363RR01
更新日期:2004-03-01 00:00:00
abstract::The Sequence Analysis Workshop (SAW) is an interactive program for sequence analysis of immunoglobulin variable domains. Sequences for SAW can be obtained from GenBank or from a standard text file. SAW can compare a variable domain to as many as 100 different sequences, calculate the extent of homology, sort the seque...
journal_title:BioTechniques
pub_type:
doi:
更新日期:1993-12-01 00:00:00
abstract::A head-to-tail trimer of the SV40 Bcl I-Bam H1 DNA fragment, specifying polyadenylation of RNA transcripts, was cloned as a cassette flanked by multiple restriction sites. Insertion of the trimer into several expression vectors efficiently prevented spurious expression of reporter genes resulting from transcriptional ...
journal_title:BioTechniques
pub_type: 杂志文章
doi:
更新日期:1989-03-01 00:00:00
abstract::Replication studies on human immunodeficiency virus 1 (HIV-1) rely on a few laboratory strains that are divergent from dominant HIV-1 subtypes in the epidemic. Several phenotypic differences between diverse HIV-1 isolates and subtypes could affect vaccine development and treatment, but this research field lacks robust...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/000113119
更新日期:2009-05-01 00:00:00