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290 records returned

Chaotic micromixing in open wells using audio-frequency acoustic microstreaming.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Mixing fluids for biochemical assays is problematic when volumes are very small (on the order of the 10 microL typical of single drops), which has inspired the development of many micromixing devices. In this paper, we show that micromixing is possible in the simple open wells of standard laboratory consumables using appropriate acoustic frequencies that can be applied using cheap, conventional audio components. Earlier work has shown that the phenomenon of acoustic microstreaming can mix fluids, provided that bubbles are introduced into a specially designed microchamber or that high-frequency surface acoustic wave dev...
Source: BioTechniques - October 1, 2009 Category: Biotechnology Authors: Petkovic-Duran K, Manasseh R, Zhu Y, Ooi A Tags: Biotechniques Source Type: journals

Heterogeneous transition metal-based fluorescence polarization (HTFP) assay for probing protein interactions.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Analyses of protein interactions are fundamental for the investigation of molecular mechanisms responsible for cellular processes and diseases, as well as for drug discovery in the pharmaceutical industry. The present study details the development of a fluorescence polarization assay using melanoma inhibitory activity (MIA) protein-binding compounds and studies of the binding properties of this protein. Since they are dependent on the the lifetime of the fluorescent label, currently available fluorescence polarization assays can only determine interactions with either high- or low-molecular weight interaction partners....
Source: BioTechniques - October 1, 2009 Category: Biotechnology Authors: Riechers A, Schmidt J, König B, Bosserhoff AK Tags: Biotechniques Source Type: journals

A dual-activation, adenoviral-based system for the controlled induction of DNA double-strand breaks by the restriction endonuclease SacI.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Spontaneous damage to DNA is frequent and may lead to cell death, cell senescence, or mutations. DNA double-strand breaks (DSBs) are of special interest because they are highly toxic and have been implicated in neurodegeneration, cancer, and aging. Until now, there has not been a reliable system allowing tunable induction of random DSBs without affecting other macromolecules or cell functions. Here, we describe an adenoviral-based, doxycycline-mediated, and tamoxifen-dependent system for quantitative introduction of DSBs in mammalian cells. We generated a single adenoviral vector containing a tet-inducible, composite S...
Source: BioTechniques - October 1, 2009 Category: Biotechnology Authors: Maslov AY, Metrikin M, Vijg J Tags: Biotechniques Source Type: journals

Reducing the impact of PCR-mediated recombination in molecular evolution and environmental studies using a new-generation high-fidelity DNA polymerase.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
PCR-mediated recombination can greatly impact estimates of diversity, both in environmental studies and in analyses of gene family evolution. Here we measure chimera (PCR-mediated recombinant) formation by analyzing a mixture of eight partial actin sequences isolated from the amoeba Arcella hemisphaerica amplified under a variety of conditions that mimic standard laboratory situations. We further compare a new-generation proofreading processivity-enhanced polymerase to both a standard proofreading enzyme and previously published results. Proofreading polymerases are preferred over other polymerases in instances where e...
Source: BioTechniques - October 1, 2009 Category: Biotechnology Authors: Lahr DJ, Katz LA Tags: Biotechniques Source Type: journals

Using phiX174 DNA as an exogenous reference for measuring mitochondrial DNA copy number.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 efficiency of nDNA and mtDNA and variation in the ploidy of experimental samples. Here, we report that the ratio of mtDNA to nDNA varies in repeated DNA extractions but that PhiX174 DNA, added before DNA extraction, is extracted with a similar efficiency to mtDNA, making it a suitable alternative reference...
Source: BioTechniques - October 1, 2009 Category: Biotechnology Authors: Myers MB, Mittelstaedt RA, Heflich RH Tags: Biotechniques Source Type: journals

A simplified, 96-well-adapted, ATP luminescence-based motility assay.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Directional motility assays make use of Boyden chambers or Transwell culture inserts with porous membranes that separate cells seeded in the upper chamber from a chemoattractant supplied in a lower chamber. These assays are often time-consuming and are associated with several limitations due to manual counting and inconsistent results; low signal-to-noise ratio and fluorescence interference; and high cost and the need for specific equipment. Here, we describe a simple, direct, and easy ATP luminescence-based motility assay (ALMA), which can be used for 96-well plate quantification. PMID: 19852771 [PubMed - in proce...
Source: BioTechniques - October 1, 2009 Category: Biotechnology Authors: Restouin A, Aresta S, Prébet T, Borg JP, Badache A, Collette Y Tags: Biotechniques Source Type: journals

Next-generation 9216-microwell cell arrays for high-content screening microscopy.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Reverse transfection on cell arrays is a high-throughput method for the parallel transfection of mammalian cells for use in high-content screening light microscopy. Here, we present novel 9216-microwell cell arrays which combine the advantages of multiwell plates (physically separated samples) and cell microarrays (high sample density and long-term storage). PMID: 19852772 [PubMed - in process] (Source: BioTechniques)
Source: BioTechniques - October 1, 2009 Category: Biotechnology Authors: Reymann J, Beil N, Beneke J, Kaletta PP, Burkert K, Erfle H Tags: Biotechniques Source Type: journals

Functional assessment of temperature-gated ion-channel activity using a real-time PCR machine.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The functional activity of a number of ion channels is highly sensitive to large changes in temperature. Foremost among these are the thermosensing TRP channels which include cold- (TRPM8, TRPA1), warmth- (TRPV3, TRPV4), and heat-sensing (TRPV1, TRPV2) members. TRPV1, also known as the vanilloid receptor (VR1), is activated by ligands such as capsaicin, acidic pH, and heat (an increase in temperature to approximately 42 degrees C will lead to channel opening). Screening against the thermal gating of TRPV1 is generally performed using perfusion systems or water baths for temperature control, in conjunction with electrop...
Source: BioTechniques - September 1, 2009 Category: Biotechnology Authors: Reubish D, Emerling D, Defalco J, Steiger D, Victoria C, Vincent F Tags: Biotechniques Source Type: journals

Arraycount, an algorithm for automatic cell counting in microwell arrays.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Microscale technologies have emerged as a powerful tool for studying and manipulating biological systems and miniaturizing experiments. However, the lack of software complementing these techniques has made it difficult to apply them for many high-throughput experiments. This work establishes Arraycount, an approach to automatically count cells in microwell arrays. The procedure consists of fluorescent microscope imaging of cells that are seeded in microwells of a microarray system and then analyzing images via computer to recognize the array and count cells inside each microwell. To start counting, green and red fluore...
Source: BioTechniques - September 1, 2009 Category: Biotechnology Authors: Kachouie N, Kang L, Khademhosseini A Tags: Biotechniques Source Type: journals

A novel method for sensitive and specific detection of DNA methylation biomarkers based on DNA restriction during PCR cycling.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
DNA methylation is an important epigenetic mechanism involved in fundamental biological processes such as development, imprinting, and carcino-genesis. For these reasons, DNA methylation represents a valuable source for cancer biomarkers. Methods for the sensitive and specific detection of methylated DNA are a prerequisite for the implementation of DNA biomarkers into clinical routine when early detection based on the analysis of body fluids is desired. Here, a novel technique is presented for the detection of DNA methylation biomarkers, based on real-time PCR of bisulfite-treated template with enzymatic digestion of b...
Source: BioTechniques - September 1, 2009 Category: Biotechnology Authors: Kneip C, Schmidt B, Fleischhacker M, Seegebarth A, Lewin J, Flemming N, Seemann S, Schlegel T, Witt C, Liebenberg V, Dietrich D Tags: Biotechniques Source Type: journals

An improved Huffman coding method for archiving text, images, and music characters in DNA.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
An improved Huffman coding method for information storage in DNA is described. The method entails the utilization of modified unambiguous base assignment that enables efficient coding of characters. A plasmid-based library with efficient and reliable information retrieval and assembly with uniquely designed primers is described. We illustrate our approach by synthesis of DNA that encodes text, images, and music, which could easily be retrieved by DNA sequencing using the specific primers. The method is simple and lends itself to automated information retrieval. PMID: 19852760 [PubMed - in process] (Source: BioTechniques)
Source: BioTechniques - September 1, 2009 Category: Biotechnology Authors: Ailenberg M, Rotstein O Tags: Biotechniques Source Type: journals

Cyclic stretch of the substratum using a shape-memory alloy induces directional migration in Dictyostelium cells.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Living cells are constantly subjected to various mechanical stimulations. They must sense the mechanical aspects of their environment and respond appropriately for proper cell function. In general, cells adhere to substrata. Thus, the cells must receive and respond to mechanical stimuli mainly from the substrata. For example, migrating cells can create their own polarity and migrate in a certain direction even in the absence of any attractive substance. In order to generate such polarity, cells must sense mechanical stimuli from the substrata and transduce these stimuli into intracellular signals. To investigate the re...
Source: BioTechniques - September 1, 2009 Category: Biotechnology Authors: Iwadate Y, Yumura S Tags: Biotechniques Source Type: journals

Direct pH measurements by using subcellular targeting of 5(and 6-) carboxyseminaphthorhodafluor in mammalian cells.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
As a means of reliably measuring intracellular pH, we have precisely targeted 5(and 6-) carboxyseminaphthorhodafluor to cellular subcompartments. This was accomplished by combining the well-established pH-sensitive dye with a protein-based reporter system. When expressed in cells, the reporter protein is designed to covalently bind ligands composed of a functional group and a reactive linker. In order to make a pH-sensitive ligand, we chemically coupled the pH sensor to a reactive linker. Several ligands of differing linker lengths were made and tested for their pH responsiveness in vitro. The most responsive of these ...
Source: BioTechniques - September 1, 2009 Category: Biotechnology Authors: Benink H, McDougall M, Klaubert D, Los G Tags: Biotechniques Source Type: journals

Enhanced amplification of GC-rich DNA with two organic reagents.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Amplification of GC-rich DNA sequences is still a difficult task worldwide. Two frequently seen and inexpensive reagents-ethylene glycol and 1,2-propanediol-were found to be more effective than betaine in the amplification of 104 randomly selected GC-rich human DNA sequences with GC contents of 60-80% and lengths of 700-800 bp. PMID: 19852763 [PubMed - in process] (Source: BioTechniques)
Source: BioTechniques - September 1, 2009 Category: Biotechnology Authors: Zhang Z, Yang X, Meng L, Liu F, Shen C, Yang W Tags: Biotechniques Source Type: journals

New clustering module in BDPC bisulfite sequencing data presentation and compilation web application for DNA methylation analyses.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The Bisulfite Sequencing Data Presentation and Compilation (BDPC) web interface for compilation and presentation of data from bisulfite sequencing DNA methylation studies has been improved by adding a new module. This module allows visualization of the whole data set in form of a heatmap of DNA methylation levels and clustering and comparison of tissue methylation patterns. It can also be used for data not processed by BDPC. In addition, several functions of the existing BDPC compilation module have been improved. PMID: 19852764 [PubMed - in process] (Source: BioTechniques)
Source: BioTechniques - September 1, 2009 Category: Biotechnology Authors: Rohde C, Zhang Y, Stamerjohanns H, Hecher K, Reinhardt R, Jeltsch A Tags: Biotechniques Source Type: journals

A rapid method for titrating baculovirus stocks using the Sf-9 Easy Titer cell line.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
A new rapid method for titrating baculovirus stocks has been developed using a novel cell line Sf-9 Easy Titer (Sf-9ET). The Sf-9ET cell line has been transfected with plasmid DNA containing the enhanced green fluorescent protein (eGFP) gene under the control of the baculovirus polyhedrin promoter. When used in the titration assay, the Sf-9ET cells turn green when they are infected with baculovirus due to the activation of the polyhedrin promoter/eGFP complex by baculovirus gene products expressed during the infection. Using a 96-well plate format end-point dilution assay, baculovirus titers can be determined in three ...
Source: BioTechniques - September 1, 2009 Category: Biotechnology Authors: Hopkins R, Esposito D Tags: Biotechniques Source Type: journals

A new outlier removal approach for cDNA microarray normalization.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Normalization is a critical step in the analysis of microarray gene expression data. For dual-labeled array, traditional normalization methods assume that the majority of genes are non-differentially expressed and that the number of overexpressed genes approximately equals the number of under-expressed genes. However, these assumptions are inappropriate in some particular conditions. Differentially expressed genes have a negative impact on normalization and are regarded as outliers in statistics. We propose a new outlier removal-based normalization method. Simulated and real data sets were analyzed, and our results dem...
Source: BioTechniques - July 31, 2009 Category: Biotechnology Authors: Wu Y, Yan L, Liu H, Sun H, Xie H Tags: Biotechniques Source Type: journals

SplinkBES: a splinkerette-based method for generating long end sequences from large insert DNA libraries.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
We report on the development of a novel splinkerette-based method for generating long end sequences from large insert library clones, using a carrot (Daucus carota L.) BAC library as a model. The procedure involves digestion of the BAC DNA with a 6-bp restriction enzyme, followed by ligation of splinkerette adaptors that serve as primer-annealing sites for PCR amplification of the BAC ends. The resulting amplicons are sequenced from both directions, and when overlapping, the pairs of sequences are assembled, originating two types of BAC end sequences (BESs): ungapped and gapped. The average sequence length for ungapped and...
Source: BioTechniques - July 31, 2009 Category: Biotechnology Authors: Cavagnaro PE, Senalik D, Simon PW Tags: Biotechniques Source Type: journals

Assessing a novel room-temperature RNA storage medium for compatibility in microarray gene expression analysis.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
RNA integrity is a critical factor in obtaining meaningful gene expression data. Current methodologies rely on maintaining samples in cold environments during collection, transport, processing, and storage procedures, which are also extremely time-sensitive. Several RNA storage products are commercially available to help prevent degradation during the handling and storage steps; however, samples must be kept cold for optimal protection. We have evaluated a novel RNA storage medium based on anhydrobiosis for stabilizing and protecting samples from degradation at room temperature that are intended for use in microarray a...
Source: BioTechniques - July 31, 2009 Category: Biotechnology Authors: Hernandez GE, Mondala TS, Head SR Tags: Biotechniques Source Type: journals

Complete discrimination of six individuals based on high-resolution melting of hypervariable regions I and II of the mitochondrial genome.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Analysis of mitochondrial DNA in forensic samples is routinely carried out by direct sequencing of hypervariable regions within the non-coding displacement loop. Although the accuracy and sensitivity of this method cannot be questioned, it is both time-consuming and labor intensive. Finding a way to rapidly pre-screen forensic samples-prior to sequencing, to reduce the number of samples that need to be sequenced-would greatly benefit forensic laboratories. Herein, we describe an assay for discrimination of DNA from different individuals based on high-resolution melting analysis of the two hypervariable regions HVI and ...
Source: BioTechniques - July 31, 2009 Category: Biotechnology Authors: Gidlöf O, Burvall S, Edvinsson L, Montelius M, Allen M, Molin M Tags: Biotechniques Source Type: journals

Chemical synthesis of oligonucleotides using acetone as a washing solvent.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Acetone is being proposed as the main washing solvent during automated synthesis of oligonucleotides to reduce acetonitrile usage in the assembly process and endure the impact of global acetonitrile shortage. PMID: 19737134 [PubMed - in process] (Source: BioTechniques)
Source: BioTechniques - July 31, 2009 Category: Biotechnology Authors: Gaytán P Tags: Biotechniques Source Type: journals

Methods comparison for high-resolution transcriptional analysis of archival material on Affymetrix Plus 2.0 and Exon 1.0 microarrays.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
This report compares transcript detection rates on Affymetrix U133 Plus 2.0 and Human Exon 1.0 ST GeneChips across several RNA extraction and target labeling protocols, using routinely collected archival FFPE samples. All RNA extraction protocols tested (Ambion-Optimum, Ambion-RecoverAll, and Qiagen-RNeasy FFPE) provided extracts suitable for microarray hybridization. Compared with Affymetrix One-Cycle labeled extracts, NuGEN system protocols utilizing oligo(dT) and random hexamer primers, and cDNA target preparations instead of cRNA, achieved percent present rates up to 55% on Plus 2.0 arrays. Based on two paired-sample a...
Source: BioTechniques - June 30, 2009 Category: Biotechnology Authors: Linton K, Hey Y, Dibben S, Miller C, Freemont A, Radford J, Pepper S Tags: Biotechniques Source Type: journals

A rapid, cost-effective method for counting human embryonic stem cell numbers as clumps.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Enumeration of human embryonic stem cell (hESC) numbers through single cell digestion can be time consuming especially in high-throughput or multi-factorial analysis containing 50+ samples. We have developed a reproducible, cost-effective method of counting hESCs in clumps circumventing the need to manually dissociate each sample to single cells. The method is based on the DNA binding capacity of propidium iodide (PI) and subsequent fluorescent signal detection. Standard curves generated for cell numbers versus PI fluorescence as single cells or clumps showed an almost identical relationship in the lines of best fit. T...
Source: BioTechniques - June 30, 2009 Category: Biotechnology Authors: Prowse A, Wolvetang E, Gray P Tags: Biotechniques Source Type: journals

Cell-free cloning using multiply-primed rolling circle amplification with modified RNA primers.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
This study demonstrates that MPRCA using random RNA primers[#x02014]instead of DNA primers[#x02014]blocked the synthesis of by-products and succeeded in amplifying one copy of a circular DNA molecule more than 1012-fold to give microgram quantities of amplification product without using submicroliter reaction volumes. Furthermore, a ligation strategy was elaborated to circularize only the desired DNA sequence and eliminate undesired ligation-products. A combination of these methods was able to amplify and ligate a large construct without undesired DNA sequences and at microgram quantities within one day. Therefore, these m...
Source: BioTechniques - June 30, 2009 Category: Biotechnology Authors: Takahashi H, Yamamoto K, Ohtani T, Sugiyama S Tags: Biotechniques Source Type: journals

Transcriptional effects of transfection: the potential for misinterpretation of gene expression data generated from transiently transfected cells.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Transfection is used to introduce a gene of interest into a cell. To interpret the downstream results, understanding which effects are the true biological responses to the gene and which, if any, are off-target effects can be difficult. In order to discriminate true biological effects from off-target effects, we transfected a breast cancer cell line, MCF7, with a vector encoding either a reporter gene or the identical vector without the reporter gene insert. Both resulted in similar numbers of differentially expressed transcripts, suggesting that very few of the responses were directly due to the introduction of the re...
Source: BioTechniques - June 30, 2009 Category: Biotechnology Authors: Jacobsen L, Calvin S, Lobenhofer E Tags: Biotechniques Source Type: journals

Bioluminescent CXCL12 fusion protein for cellular studies of CXCR4 and CXCR7.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Chemokine CXCL12 and its two known receptors, CXCR4 and CXCR7, may play a role in diseases including tumor growth and metastasis, atherosclerosis, and HIV infection. Therefore, these molecules may be promising targets for drug development. While studies of cell signaling and high-throughput screening for drug discovery increasingly are based on luminescent assays because of their high sensitivity and signal-to-background ratio, there currently is no bioluminescent assay for chemokine[#x02013]chemokine receptor binding. To develop a bioluminescent probe for chemokine binding and cellular uptake, we fused CXCL12 to Gauss...
Source: BioTechniques - June 30, 2009 Category: Biotechnology Authors: Luker K, Gupta M, Luker G Tags: Biotechniques Source Type: journals

An improved method for large-scale preparation of negatively and positively supercoiled plasmid DNA.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
A rigorous understanding of the biological function of superhelical tension in cellular DNA requires the development of new tools and model systems for study. To this end, an ethidium bromide[#x02013]free method has been developed to prepare large quantities of either negatively or positively super-coiled plasmid DNA. The method is based upon the known effects of ionic strength on the direction of binding of DNA to an archaeal histone, rHMfB, with low and high salt concentrations leading to positive and negative DNA supercoiling, respectively. In addition to fully optimized conditions for large-scale (>500 microg) s...
Source: BioTechniques - June 30, 2009 Category: Biotechnology Authors: Barth M, Dederich D, Dedon P Tags: Biotechniques Source Type: journals

Cell-free protein synthesis using multiply-primed rolling circle amplification products.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
In this study, we tested whether RCA-generated DNA can serve as the template for in vitro transcription. We found that RCA DNA[#x02013]generated transcripts work in coupled in vitro translation with nearly the same efficiency (per nanogram of DNA) as those obtained from purified plasmid. We propose a convenient, single-tube format for template amplification, transcription, and translation. Address correspondence to Gyanendra Kumar, GE Healthcare Bio-Sciences Corp. Advanced Systems Division, 800 Centennial Avenue, Piscataway, NJ, 08854, USA. email: kumargyan@hotmail.com. PMID: 19594449 [PubMed - in process] (Source: BioTechniques)
Source: BioTechniques - June 30, 2009 Category: Biotechnology Authors: Kumar G, Chernaya G Tags: Biotechniques Source Type: journals

Multiplex Manager 1.0: a cross-platform computer program that plans and optimizes multiplex PCR.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 smallest number of reactions, or alternatively, selecting a subset from many available markers to design an efficient and robust multiplex. Our program minimizes the number of reactions, the genetic linkage, and the difference in annealing temperature. At the same time it maximizes the spacing between markers, t...
Source: BioTechniques - May 31, 2009 Category: Biotechnology Authors: Holleley C, Geerts P Tags: Biotechniques Source Type: journals

Design of FRET-TaqMan probes for multiplex real-time PCR using an internal positive control.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The multiplexing capabilities with different fluorescent dyes are limited in real-time PCR instruments equipped with one excitation source. Considering this limitation, a design was developed to create a triple-labeled probe as an internal positive control (IPC) that utilizes a combination of the fluorescence resonance energy transfer (FRET) and TaqMan techniques. The IPC probe, labeled with FAM and Cy5.5 fluorophores at the 5' end and Black Hole Quencher (BHQ) at the 3' end, enabled Cy5.5 emission through energy transfer from the FAM fluorophore. The second, target-specific TaqMan assay in the multiplex used a FAM- an...
Source: BioTechniques - May 31, 2009 Category: Biotechnology Authors: Jothikumar P, Hill V, Narayanan J Tags: Biotechniques Source Type: journals

Site-directed mutagenesis of multi-copy-number plasmids: Red/ET recombination and unique restriction site elimination.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Existing methods for site-directed plasmid mutagenesis are restrained by the small spectrum of modifications that can be introduced by mutagenic primers and the amplicon size limitations of in vitro DNA synthesis. As demonstrated here, the combined use of Red/ET recombination and unique restriction site elimination enables extensive manipulation regardless of plasmid size and DNA sequence elements. First, a selectable marker is PCR-amplified with synthetic primers attaching 50-bp homology target flanks for Red/ET recombination and an arbitrary restriction site absent in the substrate plasmid. The resulting cassette is ...
Source: BioTechniques - May 31, 2009 Category: Biotechnology Authors: Noll S, Hampp G, Bausbacher H, Pellegata N, Kranz H Tags: Biotechniques Source Type: journals

Transposon-directed base-exchange mutagenesis (TDEM): a novel method for multiple-nucleotide substitutions within a target gene.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
In this report we describe transposon-directed base-exchange mutagenesis (TDEM), an efficient and controllable method for introducing a mutation into a gene. Each round of TDEM can remove up to 11 base pairs from a randomly selected site within the target gene and replace them with any length of DNA of predetermined sequence. Therefore, the number of bases to be deleted and inserted can be independently regulated providing greater versatility than existing methods of transposon-based mutagenesis. Subsequently, multiple rounds of mutagenesis will provide a diverse mutant library that contains multiple mutations throughout t...
Source: BioTechniques - May 31, 2009 Category: Biotechnology Authors: Kim YC, Lee HS, Yoon S, Morrison S Tags: Biotechniques Source Type: journals

Simple non-fluorescent polarity labeling of microtubules for molecular motor assays.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Transport of intracellular organelles along the microtubule cytoskeleton occurs in a bidirectional manner due to opposing activity of microtubule-associated motor proteins of the kinesin and dynein families. Regulation of this opposing activity and the resultant motion is believed to generate a polarized distribution of many organelles within the cell. The bidirectional motion can be reconstituted on in vitro assembled microtubules using organelles extracted from cells. This provides an opportunity to understand the regulation of intracellular transport through quantitative analysis of the motion of organelles in a con...
Source: BioTechniques - May 31, 2009 Category: Biotechnology Authors: Soppina V, Rai A, Mallik R Tags: Biotechniques Source Type: journals

Mouse embryo cryopreservation utilizing a novel high-capacity vitrification spatula.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Embryo cryopreservation is an indispensable technique in reproductive programs and in animal facilities where genetically modified mice are used extensively. Here we report the use of a vitrification spatula (VS) that can be readily homemade and has a large holding capacity to vitrify preimplantation mammalian embryos in a micro-drop employing ultra-rapid cooling in liquid nitrogen (LN2). Vitrified one-cell embryos and morulae have high survival rates after thawing, and the fertility of the derived progeny is comparable to that of the control unvitrified group. The large holding capacity (up to 50 embryos per VS) does ...
Source: BioTechniques - May 31, 2009 Category: Biotechnology Authors: Tsang WH, Chow K Tags: Biotechniques Source Type: journals

A device for automated hydrodynamic shearing of genomic DNA.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
We describe a device for automated fragmentation of genomic DNA by hydrodynamic shearing using a filter screen with uniform pores. Human genomic DNA can be fragmented reproducibly to a range of 2000-12,000 bp by using various fluid flow rates and screens with 0.5-10-microm pores. The utilization of disposable screens eliminates sample cross-contamination and the tendency of device clogging, commonly encountered in single-orifice shearing devices. Address correspondence to Xiaohua Huang, Department of Bioengineering University of California, San Diego, 9500 Gilman Drive, La Jolla, CA, 92093, USA. e-mail: x2huang@ucsd.edu. ...
Source: BioTechniques - May 31, 2009 Category: Biotechnology Authors: Joneja A, Huang X Tags: Biotechniques Source Type: journals

Analysis of insoluble proteins.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The analysis of insoluble proteins represents a major technical challenge for the field of proteomics. For example, membrane proteins are often insoluble in common solvents and represent 20-30% of the proteins encoded by the human genome. Chemical analysis on an individual basis is often required and is laborious and time-consuming. This review presents an overview of methods for purification of expressed proteins using fusion tags as well as methods for analysis of insoluble proteins by mass spectrometry with a goal of achieving high-throughput analysis. PMID: 19480635 [PubMed - in process] (Source: BioTechniques)
Source: BioTechniques - May 1, 2009 Category: Biotechnology Authors: Trimpin S, Brizzard B Tags: Biotechniques Source Type: journals

Prim-SNPing: a primer designer for cost-effective SNP genotyping.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Many kinds of primer design (PD) software tools have been developed, but most of them lack a single nucleotide polymorphism (SNP) genotyping service. Here, we introduce the web-based freeware "Prim-SNPing," which, in addition to general PD, provides three kinds of primer design functions for cost-effective SNP genotyping: natural PD, mutagenic PD, and confronting two-pair primers (CTPP) PD. The natural PD and mutagenic PD provide primers and restriction enzyme mining for polymerase chain reaction-restriction fragment of length polymorphism (PCR-RFLP), while CTPP PD provides primers for restriction enzyme-free SNP genot...
Source: BioTechniques - May 1, 2009 Category: Biotechnology Authors: Chang HW, Chuang LY, Cheng YH, Hung YC, Wen CH, Gu DL, Yang CH Tags: Biotechniques Source Type: journals

Cell cycle quiescence can suppress transcription from an ecdysone receptor-based inducible promoter in mammalian cells.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Inducible gene expression is a powerful tool for basic research, gene therapy and biotechnology, whose utility depends in part on consistent levels of induction regardless of metabolic status or physiological context. Here we examined the inducibility of the ecdysone receptor-based RheoSwitch mammalian inducible expression system in proliferating cells and in cell cycle-arrested cells. We found that both contact inhibition and growth arrest subsequent to serum deprivation dramatically reduced the levels of induction of reporter genes that could be achieved in 3T3 fibroblasts but in not NMuMG mammary epithelial cells. T...
Source: BioTechniques - May 1, 2009 Category: Biotechnology Authors: Wallbaum S, Grau N, Schmid A, Frick K, Neeb A, Sleeman JP Tags: Biotechniques Source Type: journals

ProReg XL Tool: an easy-to-use computer tool suite for rapidly regrouping a large number of identical electrophoretic profiles.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The ProReg XL Tool (Profile Regrouping Excel Tool) is a new tool suite designed to rapidly regroup a large number of identical electrophoretic profiles. This tool suite is coded in Visual Basic Application for Microsoft Excel, and thus requires this spreadsheet software to operate. It was designed for use with a new screening strategy of clones from an rrs (16S rDNA) clone library, but it may also be helpful in other electrophoretic applications. ProReg XL Tool is organized in different steps where the user has the capability--in addition to regrouping electrophoretic profiles--to control gel quality, determine signal ...
Source: BioTechniques - May 1, 2009 Category: Biotechnology Authors: Massias B, Urdaci MC Tags: Biotechniques Source Type: journals

Positive-selection vector for direct protein expression.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
We describe the development of a novel positive-selection vector, RHP-AmpS, that is suitable for seamless cloning and high-level protein expression in Escherichia coli. In this vector, beta-lactamase (Bla) was rendered nonfunctional by replacing the codon for the C-terminal amino acid of the beta-lactamase gene (bla) with a stop codon. Insertion of a target gene in the correct orientation (tail to tail) results in the reconstruction of the C-terminal codon (W290) of bla. This restores the function of the gene and allows the selection of positive recombinants on agar plates containing ampicillin. To allow a high level of pr...
Source: BioTechniques - May 1, 2009 Category: Biotechnology Authors: Haag AF, Ostermeier C Tags: Biotechniques Source Type: journals

A novel yeast-based recombination method to clone and propagate diverse HIV-1 isolates.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
In this study, we describe an alternative approach that involves direct introduction of any HIV-1 coding regions (e.g., any gene from a patient sample) into an HIV-1 DNA vector using yeast recombination. This technique uses positive and negative selectable markers in yeast and avoids the need for purification and screening of the DNA substrates and cloning products. Replication-competent virus is then produced from a modified mammalian 293T packaging cell line transfected with this yeast-derived HIV-1 vector. Although HIV-1 served as the prototype, this cloning strategy is now being developed for other diverse virus specie...
Source: BioTechniques - May 1, 2009 Category: Biotechnology Authors: Dudley DM, Gao Y, Nelson KN, Henry KR, Nankya I, Gibson RM, Arts EJ Tags: Biotechniques Source Type: journals

Pressure: a novel tool for enzyme-linked immunosorbent assay procedure.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The enzyme-linked immunosorbent assay (ELISA) technique is the backbone of the diagnostic assay for detection of infectious and allergic diseases. Here, we demonstrate a unique ELISA method for the detection of an antigen or antibody, in which ELISA steps are carried out under pressure instead of conventional thermal incubation. Pressure-mediated ELISA (PELISA), carried out in 1 h shows more than a 2-fold increase in absorbance value than the control experiment carried out at the same time and temperature without applying pressure. Estimation of total IgE by the 1-h PELISA method gives similar absorbance value (1.081+/...
Source: BioTechniques - May 1, 2009 Category: Biotechnology Authors: Kannoujia DK, Nahar P Tags: Biotechniques Source Type: journals

Nanoliter high-throughput RT-qPCR: a statistical analysis and assessment.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Biomarkers discovered from gene expression profiles using hybridization microarrays have made great inroads in the diagnosis and development of safer and efficacious drugs. The candidate gene set is biologically validated by quantitative measurement with reverse transcriptase quantitative PCR (RT-qPCR) and is an effective strategy when implemented with microplates if the number of candidate genes and samples is small. With the trend toward informative candidate gene panels increasing from tens to hundreds of genes and sample cohorts exceeding several hundred, an alternative fluidic approach is needed that preserves the...
Source: BioTechniques - May 1, 2009 Category: Biotechnology Authors: Dixon JM, Lubomirski M, Amaratunga D, Morrison TB, Brenan CJ, Ilyin SE Tags: Biotechniques Source Type: journals

A versatile multidimensional protein purification system with full Internet remote control based on a standard HPLC system.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The standard Akta Explorer high-performance liquid chromatography (HPLC) system has limitations for the automation of multidimensional protein purification. Here, we describe simple modifications that allow for automated multidimensional purification protocols to extend the possibilities of the Akta three-dimensional purification kit in terms of column number, flexibility of volumes stocked for re-injection of samples, and available choice of buffers. These modifications do not preclude the use of standard one-dimensional purification protocols. Additionally, we demonstrate a technology for encrypted full remote contro...
Source: BioTechniques - May 1, 2009 Category: Biotechnology Authors: Riek U, Ramirez S, Wallimann T, Schlattner U Tags: Biotechniques Source Type: journals

TScratch: a novel and simple software tool for automated analysis of monolayer wound healing assays.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Cell migration plays a major role in development, physiology, and disease, and is frequently evaluated in vitro by the monolayer wound healing assay. The assay analysis, however, is a time-consuming task that is often performed manually. In order to accelerate this analysis, we have developed TScratch, a new, freely available image analysis technique and associated software tool that uses the fast discrete curvelet transform to automate the measurement of the area occupied by cells in the images. This tool helps to significantly reduce the time needed for analysis and enables objective and reproducible quantification o...
Source: BioTechniques - April 1, 2009 Category: Biotechnology Authors: Gebäck T, Schulz MM, Koumoutsakos P, Detmar M Tags: Biotechniques Source Type: journals

A novel L1 retrotransposon marker for HeLa cell line identification.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The HeLa cell line is the oldest, most widely distributed, permanent human cell line. As a nearly ubiquitous inhabitant of laboratories using tissue culture techniques, its aggressive growth characteristics make it a problematic contaminant that can overgrow less robust cell lines. Consequently, HeLa contamination is common in both the research laboratory and cell line repository contexts, and its detection is hampered by the lack of a rapid, sensitive and robust assay. Here we report the development of a HeLa-specific DNA diagnostic test: a single duplex detection PCR assay targeting an L1 retrotransposon insertion. A...
Source: BioTechniques - April 1, 2009 Category: Biotechnology Authors: Rahbari R, Sheahan T, Modes V, Collier P, Macfarlane C, Badge RM Tags: Biotechniques Source Type: journals

Thin-sheet laser imaging microscopy for optical sectioning of thick tissues.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
We report the development of a modular and optimized thin-sheet laser imaging microscope (TSLIM) for nondestructive optical sectioning of organisms and thick tissues such as the mouse cochlea, zebrafish brain/inner ear, and rat brain at a resolution that is comparable to wide-field fluorescence microscopy. TSLIM optically sections tissue using a thin sheet of light by inducing a plane of fluorescence in transparent or fixed and cleared tissues. Moving the specimen through the thinnest portion of the light sheet and stitching these image columns together results in optimal resolution and focus across the width of a large sp...
Source: BioTechniques - April 1, 2009 Category: Biotechnology Authors: Santi PA, Johnson SB, Hillenbrand M, GrandPre PZ, Glass TJ, Leger JR Tags: Biotechniques Source Type: journals

Subcellular protein extraction from human pancreatic cancer tissues.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Proteins are the major class of effector molecules in cellular systems. For the identification of functional differences between normal and diseased tissues, a reliable analysis of their protein content is essential. Reproducible isolation and fractionation of intact proteins are important in this respect, but their complexity in structure and concentration, their close interaction, and their instability represent major challenges. For protein isolation in tissues, the breakdown of cell-cell and cell-matrix connections within a tissue without affecting protein quality is a critical factor. We compared different process...
Source: BioTechniques - April 1, 2009 Category: Biotechnology Authors: Börner A, Warnken U, Schnölzer M, Hagen J, Giese N, Bauer A, Hoheisel J Tags: Biotechniques Source Type: journals

An agarose-based cloning-ring anchoring method for isolation of viable cell clones.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Isolation of clonal cell populations is a crucial aspect of cell biology during engineering of specific cell strains with both genotypic and phenotypic variations. The use of cloning rings is the most established method, but requires anchoring chemicals or material that can often interfere with quantitative clonal-cell isolation and causes physical damage to the cells. Here we report a non-toxic, cell culture-compatible method that uses aga-rose for embedding the cloning rings during isolation of cell clones from monolayer cultures, with enhanced cell-viability and reproducibility during downstream applications. The me...
Source: BioTechniques - April 1, 2009 Category: Biotechnology Authors: Mathupala S, Sloan AA Tags: Biotechniques Source Type: journals

DNA elution from buccal cells stored on Whatman FTA Classic Cards using a modified methanol fixation method.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
We describe here a method for DNA elution from buccal cells and whole blood both collected onto Whatman FTA technology, using methanol fixation followed by an elution PCR program. Extracted DNA is comparable in quality to published Whatman FTA protocols, as judged by PCR-based genotyping. Elution of DNA from the dried sample is a known rate-limiting step in the published Whatman FTA protocol; this method enables the use of each 3-mm punch of sample for several PCR reactions instead of the standard, one PCR reaction per sample punch. This optimized protocol therefore extends the usefulness and cost effectiveness of each buc...
Source: BioTechniques - April 1, 2009 Category: Biotechnology Authors: Johanson HC, Hyland V, Wicking C, Sturm RA Tags: Biotechniques Source Type: journals