Why a microfluidic approach for continuous measurements of biofilm viscosity

A microfluidic method combining video tracking with a semi-empirical viscous flow model provides continuous, non-intrusive measurements of time-varying viscosity in biofilms from Pseudomonas sp. bacteria. The approach uses measured velocity and height of tracked biofilm segments, which move under the low shear force imposed by flowing nutrient solutions.

The initial measured viscosities for the first 24 hours after inoculation were among the lowest reported to date. Following a low viscosity growth stage, sudden thickening was observed. During this stage, viscosity increased by over an order of magnitude in less than ten hours. The technique was also demonstrated as a promising platform for parallel experiments by subjecting multiple biofilm-laden microchannels to nutrient solutions containing NaCl in the range of 0 mM to 34 mM. Even in this narrow range of ionic strengths, preliminary data suggest a strong relationship between ionic strength and biofilm properties, such as average viscosity and time of onset of rapid thickening. The technique opens the way for a combinatorial approach to study the response of biofilm viscosity under well-controlled physical, chemical and biological growth conditions.

Understanding Constant stretching of DNA in a microfluidic device

Genome Sequence Scanning (GSS) detects sequence-specific fluorescent tags on DNA fragments. It is used to identify bacteria by first lysing open the bacteria cell to release the DNA, then using a restriction endonuclease enzyme to digest the DNA into smaller fragments. Fluorescent tags are added that recognise specific repeated elements present in all bacterial genomes. GSS characterises the bacterial genome by the spatial distribution of the tags.

The detection is done in a continuous flow microfluidic device with confocal microscopy. In order to carry out the spatial recognition of the fluorescent tags along the length of the DNA fragment, it needs to be stretched out into a linear form using a funnel. High molecule throughput is important as the detection confidence of this technology relies on observing as many tags as possible in the specified experimental period.

The team looks at the relationship between the funnel taper shape and related parameters, such as fluid velocity and fragment length, to improve the current designs and increase throughput. Their new geometries are able to keep the tension applied to the DNA constant during the detection process. Because DNA fragments come in various lengths, a very important goal is to maximise the range of lengths that can be stretched effectively with the funnel. The influence of channel etch depth on fluid flow, and therefore throughput, is also considered.

what is Recombinant antibodies?

Recombinant antibodies (rAbs) are monoclonal antibodies which are generated in vitro using synthetic genes. Unlike monoclonal antibodies (mAbs) which are produced using traditional hybridoma-based technologies, rAbs do not need hybridomas and animals in the production process.

Both monoclonal and recombinant antibodies can be used in biomedical science and toxicological research, and are effective therapeutic treatments for cancer, autoimmune disorders and a host of other diseases. However, while monoclonal antibodies have become one of the most common tools in biomedical science and medicine due to their ability to bind and neutralize or destroy cell-specific antigens, the ascites method of production causes significant pain and discomfort to the animals used in the process.

As such, the governments of Australia, Germany, the Netherlands and the United Kingdon banned it in favor of in vitro methods. The US also endorses the use of in vitro methods as the default procedure for the production of mAbs.

However, it is important to note that in vitro methods involving hybridomas also have their own limitations, which include the following:

Require immunization and subsequent euthanasia of the animals used in the process.
Slow and labor-intensive.
Often cause immune reactions so the antibodies need to be altered and "humanized" before they can be administered to patients.

You need to know How do you prepare your phosphate buffer?

Since biological reactions are extremely sensitive to pH changes, it’s always a good idea to have a phosphate buffer ready in the laboratory. In case you aren’t familiar with them, phosphate buffers are one of the two most important biological buffer systems in most multicellular organisms and are responsible for keeping the pH range of cellular fluid within the normal range (6.9 to 7.4).Since phosphoric acid has multiple dissociation constants, you can prepare phosphate buffers near pH 2.15, 6.86 and 12.32. However, most phosphate buffers are prepared at pH 7.

Preparing Your Phosphate Buffer
How do you prepare your phosphate buffer? Here's how:

What You'll Need

Monosodium phosphate, monohydrate and its conjugate base disodium phosphate, heptahydrate
Water
Phosphoric acid (to make the buffer more acidic) or sodium hydroxide (to make the solution more basic)
pH meter, glassware and hot plate with stirring bar
Procedure:

Decide on the properties of your buffer. Before doing anything, you need to decide on the molarity and pH of your buffer. Remember, most buffers are used at a concentration between 0.1 M and 10 M and the pH should ideally be within 1 pH unit of the acid or its conjugate base.

Prepare the solution. Using the calculated ratioderived from the equation, mix the required amounts of acid and base and mix them in approximately one liter of water to prepare a rough buffer solution.
Check the pH. Use your pH meter and adjust the pH accordingly by using phosphoric acid or sodium hydroxide. Bring the total volume to one liter once you have reached the desired pH.
Dilute as necessary. Use this stock solution to prepare buffers of different molarities as needed.

Understanding PathoGenetix Delivers Bacterial Identification System to FDA

PathoGenetix has delivered an early commercial version of the RESOLUTION™ Microbial Genotyping System to the U.S. Food and Drug Administration (FDA). The FDA is leasing the RESOLUTION System as part of a three-year collaborative agreement to evaluate the rapid bacterial identification technology for use in FDA foodborne illness outbreak investigation and response.

The RESOLUTION System, based on PathoGenetix’s proprietary Genome Sequence Scanning™ (GSS™) technology, enables pathogen serotype identification and strain typing in just five hours, directly from complex mixtures such as enriched food and clinical samples. The bacterial strain information provided by the RESOLUTION System is comparable to pulsed field gel electrophoresis (PFGE), the current gold standard for pathogen typing in foodborne illness outbreak investigation and response.

Identifying the pathogen strain that is causing a foodborne illness outbreak is a critical step in defining the extent of the outbreak, determining the food involved, finding the original source of the contamination and defining the scope of a product recall. The ability of the RESOLUTION System to derive useful pathogen strain and serotype information directly from a complex mixture, and to shorten the time for pathogen typing to just five hours, could allow for quicker decisions affecting public health.

Current microbial identification techniques such as PFGE and whole genome sequencing (WGS) require a cultured isolate as input, and advanced, time-consuming laboratory processes for preparation and processing of food samples. Analysis of the patterns created by PFGE, or the extensive data generated by WGS, can be complex and add significantly to the time required to identify the pathogen strain and serotype. Because the PathoGenetix system is culture independent, and fully automated from sample preparation to final report, it has the potential to greatly reduce the time, complexity and skill-level required to identify foodborne pathogens in hospital and public health labs monitoring foodborne outbreaks.

Understanding How to extract biologically active proteins from the cells

Extraction of proteins from cells and tissue of organisms is the first step towards isolation of proteins. The extracellular matrix needs to be removed or digested in case of tissue, the cell wall needs to be digested for organisms like bacteria, yeast and plants, and the cell membrane needs to be disrupted to release the proteins in solution. Traditionally, physical methods for disruption of cells and tissues are employed to release cellular proteins including sonication, french press, homogenization, manual grinding or using blenders.  Although one is able to get the active proteins, these physical methods have several limitations:

• Expensive
• Cumbersome due to use of heavy equipment
• Reduced yield as the sample is processed through several steps
• Batch to batch variation in protein yield due variability and handling

Mechanical methods can sometimes denature proteins as these methods are harsh and some of them generate heat and can denature protein if appropriate cooling of sample is not done.

Chemical methods for cell disruption using lysis buffers with ionic detergents can be used to release proteins; however they can denature the protein.  Non-ionic detergents offer advantage over lysis buffer and ionic detergents as they are mild and the proteins are not denatured. Therefore, nowadays popular methods for extraction of biologically active proteins from cells or tissues use non-ionic detergents along with other additives, such as enzymes for cell wall disruption and addition of protease inhibitors to inhibit proteases. This method may involve mild mechanical methods, including brief homogenizing or vortexing depending upon the cell or tissue type.

So why PathoGenetix Announces $10 Million Series C Financing

The GSS technology confirms and identifies microbial DNA in either complex mixtures or isolates, and provides actionable information in five hours. The funding will be used to further develop the first commercial system using the GSS technology, the RESOLUTION™ Microbial Genotyping System. The fully automated RESOLUTION System includes the instrument, bioinformatics software and database, and pathogen-specific assays, and will be commercially available in 2014 for use in food safety testing and foodborne illness outbreak investigations.

“This commitment from our investors reflects the outstanding progress we have made toward commercialization of the GSS technology," said Ann Merrifield, President and CEO of PathoGenetix. "As we continue our efforts to deliver the RESOLUTION System for rapid pathogen strain typing, we also are gaining excellent market traction in the key public health and food industry sectors.”

The company announced collaborations with the Food and Drug Administration (FDA) in April and the U.S. Department of Agriculture’s Agricultural Research Service (USDA-ARS) in September, and is currently conducting a series of customer evaluations with leading food safety testing labs. In June, the company signed an agreement with Applied Maths, NV, to link the RESOLUTION System with the BioNumerics software suite currently in use in thousands of public and private research sites and laboratories around the world.

The company announced collaborations with the Food and Drug Administration (FDA) in April and the U.S. Department of Agriculture’s Agricultural Research Service (USDA-ARS) in September, and is currently conducting a series of customer evaluations with leading food safety testing labs. In June, the company signed an agreement with Applied Maths, NV, to link the RESOLUTION System with the BioNumerics software suite currently in use in thousands of public and private research sites and laboratories around the world.

You know PathoGenetix Delivers Bacterial Identification System to MRIGlobal for Evaluation

PathoGenetix™, Inc., developer of an automated system for rapid bacterial strain typing, announced today that it has delivered and installed an early commercial version of the RESOLUTION™ Microbial Genotyping System to MRIGlobal, an independent contract research organization. MRIGlobal has purchased the RESOLUTION System as part of a U.S. government-funded project, and will be evaluating use of the System for identification and strain typing of specific organisms using MRI-developed assays.

The RESOLUTION System is based on PathoGenetix’s proprietary Genome Sequence Scanning™ (GSS™) technology, which enables pathogen serotype identification and strain typing in just five hours, directly from complex mixtures such as environmental, clinical and enriched food samples. Initially developed to detect bio-threat pathogens in environmental samples under a five-year, $50-million contract through the Department of Homeland Security, the breakthrough GSS technology isolates and analyzes DNA direct from complex mixtures—without the need for a pure culture isolate. The strain type information provided by GSS is comparable in resolution to pulsed field gel electrophoresis (PFGE), one of the current gold standards for pathogen identification.

MRIGlobal and PathoGenetix have collaborated for several years in the ongoing evaluation of the GSS technology for biodefense applications. MRIGlobal has purchased a RESOLUTION System as part of a U.S. government-funded project, and will be evaluating the system for use in the rapid identification of specific organisms, using MRIGlobal-developed assays. The RESOLUTION instrument will be used to build a database for identification of select microorganisms. As one of the nation’s leading research institutes, MRIGlobal conducts programs in the areas of national security and defense, life sciences, energy and the environment, agriculture and food safety, and engineering and infrastructure.

How Do Detergents Work?

A wide range of detergents are routinely used to release, or solubilize, proteins from lipid membranes. 
Biological membranes consist of phospholipids that are similar to detergents as they have the same amphipathic properties. The phospholipids have a charged polar head normally connected to two hydrophobic groups or tails. The phospholipids assemble as bilayers, with the hydrophobic tails between two faces of polar head groups.

The proteins are released from lipid bilayers by detergents as the detergent micelles have similar properties as the lipid bilayer. The integral membrane proteins embed themselves in the detergent micelles protecting their hydrophobic domains from aggregation.
A schematic of how detergents solubilize membrane proteins is shown below. At low detergent concentrations, less than the detergent’s CMC, the detergent molecules insert themselves in the lipid membrane and begin partioning the lipid bilayer. At concentrations equal to, or higher than the detergent’s CMC, the lipid bilayer becomes saturated with detergent molecules and the lipid bilayer breaks apart. The resulting products are protein-detergent complexes, where the detergent hydrophobic regions bind to the protein hydrophobic domains protecting them from aggregations. In addition to these, detergent and detergent-lipid micelles are formed.

The manufacturing of commercial detergents, including non-ionic detergents, results in the presence of sulfhydryl oxidizing agents, peroxides, salts and carbonyl compounds.  The level of these harmful reagents, particularily the peroxides and carbonyls (aldehydes) can increase over time as the product degrades.

The graph below shows a comparison between the aldehyde and peroxidelevels in G-Biosciences Proteomic Grade Detergents and commercial non proteomic grade detergents. 

The oxidizing agents, peroxides and aldehydyes will react with protein amino acids causing changes to its primary structure, molecular mass and may also inhibit protein:protein interactions.

How does SMALL RNA SEQUENCING works?

Non-coding small RNAs (ncRNA, smallRNA) and microRNAs (miRNA) are key regulators of gene expression, heterochromatin formation and nucleus organisation. Their action is dependent on tissue and environment and, through various mechanisms, they have a major influence on cancer and numerous diseases.

Several platforms within the France Génomique network will accept requests for small RNA studies or microRNA studies (Pasteur, Strasbourg, Montpellier, Nice, Paris centre, CEA/IG-CNG).
JPEG - 39 kb
Starting with total RNA, the small RNAs are purified on acrylamide gel and used to prepare sequencing libraries. In this step, a sequence specific adapter is added to both ends of the RNA molecule, RNA is converted into double-stranded cDNA, and amplification is performed. The libraries are then quality controlled on a Bioanalyser (Agilent) and quantified on a QuBit (Invitrogen). The resulting sequences will be of native RNA (directional RNA-Seq).

What is METHYLATED DNA IMMUNOPRECIPITATION?

Video is Here 

DNA methylation is a phenomenon implicated in numerous diseases, the most frequently studied of which is cancer. In mammals, methylation occurs at cytosine bases which are followed by a guanine base (CpG), principally in the form of 5-methylcytosine:

MeDIP is a technique that uses anti-methylcytosine antibodies which are coupled to magnetic beads to immunoprecipitate the regions that are sufficiently methylated.

JPEG - 20.3 kbSample DNA is fragmented by sonication then antibodies are added. Once these have bonded to the methylcytosines, the methylated fragments are collected using a magnet, thus removing from the sample any unmethylated or weakly methylated fragments: the sample has thus been enriched with methylated sequences and is ready to be analysed using a sequencing technique. For better reproducibility, it is recommended to use a robot to perform MeDIP.

The French National Genotyping Centre (CNG), which is part of the Institute of Genomics at the CEA in Evry (near Paris), and the Toulouse platform, both have the necessary equipment to optimise this pre-sequencing step.

What is Benefits of DNA fingerprinting

Benefits in Paternity Tests

The sections of DNA used in DNA fingerprinting (as with all DNA) are passed down from parents to children. By comparing large numbers of these DNA microsatellites it is possible to establish with a high degree of certainty the paternity, maternity or both of a child.In some cases men or women need to gain peace of mind regarding their relationship to a child. A mother may have had sexual encounters with more than one man and does not know which one is the parent of her child. In other cases men may suspect the child is not his so may use DNA profiling as a way of being completely sure. Adopted children may want to know who their biological parents are. DNA testing may allow them to advance in this regard.

Also, DNA profiling could assist mothers seeking payment of alimony by a father who refuses to pay it saying he is not the child’s father. Or some people may use DNA fingerprinting to establish a link to grandparents to collect an inheritance or to claim social security benefits.

Benefits in Agriculture and Other Areas

Although this technology is mainly used in forensics and paternity tests, there are many other areas where it is being successfully applied.

In agriculture DNA fingerprinting is increasingly being used for variety identification--that is, to know if a seed is really from the variety being claimed. Also, genetic profiling is utilized to detect. More specifically, markers have been used for characterization and determination of genetic diversity of tea to find genetic relatedness. Interestingly, and with the advent of herbal preparations and alternative medicine, DNA fingerprinting of herbal preparations could be useful in herbal origin authentication, and for the various claims of medical uses related to some of these plants.

What is EpiMark Methylated DNA Enrichment Kit

The EpiMark® Methylated DNA Enrichment Kit enables the enrichment of double-stranded CpG methylated DNA based on CpG methylation density. It utilizes the methyl-CpG binding domain of human MBD2a protein as a capture agent. The protein is fused to the Fc tail of human IgG1 (MBD2a-Fc), which is coupled to Protein A Magnetic Beads (MBD2a-Fc/Protein A Bead). This stable complex will selectively bind double-stranded methylated CpG containing DNA. The high binding affinity of the beads coupled with optimized reagents increases sensitivity and accuracy. This kit contains all the individual components necessary to achieve enrichment in less than two hours using a four step process.

You now SNP Genotyping by Fragment Analysis?

SNP genotyping identifies single nucleotide polymorphisms (SNPs) that are common DNA variants present across the human genome. SNPs have been shown to be responsible for differences in genetic traits, susceptibility to disease, and response to drug therapies.

Genotyping of SNPs has become extremely important to researchers working to understand and treat disease. SNPs occur approximately once every 100 to 300 bases and can be detected by various different techniques such as RFLP, SSCP, sequencing, and more.

Go to the Publications & Literature tab below to learn some of the many ways our customers use the SNaPshot®  Multiplex System for their research.

Amazing SNP Genotyping & Variant Detection by Sequencing

Variant Detection by Sanger Sequencing
Sanger sequencing is the gold-standard sequencing technology, making it ideal for confirmation of novel variants. Designing sequencing experiments for variant detection is easier than ever with our new Primer Designer™ tool for Sanger sequencing (currently available in US & Canada), containing >300,000 predesigned primer pairs for PCR and sequencing.

Variant Detection by Ion Torrent™ Next-Generation Sequencing
With industry-leading performance and gold-standard accuracy, Sanger sequencing with Applied Biosystems® genetic analyzers serve as your trusted partner for confirming NGS results.

What is Ion semiconductor sequencing

Now, more laboratories can adopt powerful next-generation sequencing technology to get answers faster —with increased throughput, higher accuracy, and the simplest workflow from sample preparation through to data analysis. With the Ion Torrent™ Personal Genome Machine® (PGM) System, human disease researchers can detect variants by targeted gene sequencing in cancer and genetic disorders, and microbiologists can easily type a bacteria or virus, or characterize novel microbes.

The combination of Ion AmpliSeq™ target technology, an Ion PGM™ System, and fully automated template preparation from the upcoming Ion Chef™ System**, offers the simplest and fastest* workflow for gene panel sequencing. Get meaningful results in just half the time of the alternative, with highly multiplexed Ion AmpliSeq™ DNA panels, the walk away solution for preparing sequence-ready chips from libraries provided by the Ion Chef™ System, and faster sequencing on the Ion PGM™ System. From sample to results, there are no centrifugation steps, and half the pipetting steps and hands on time of the alternative NGS

You should know Sequencing Kits & Reagents

Life Technologies provides comprehensive sequencing kits and reagents developed specifically for each sequencing platform to enable the most efficient and effective sequencing results.

Sanger sequencing kits and reagents support PCR amplification of region(s) of interest, cycle sequencing, and reaction cleanup. Next-generation sequencing kits and reagents are available for target selection, library construction, and template preparation. Browse the sections below to learn more about sequencing kits and reagents to prepare samples for each of our sequencing platforms.

Next-Generation Sequencing Kits & Reagents
Browse library construction, template preparation, and sequencing kits for your Ion Torrent next-generation sequencing applications on the Ion PGM System or Ion Proton System.

Sanger Sequencing Kits & Reagents
Learn about our kits and reagents that have been optimized for Sanger sequencing workflows, including sample purification, predesigned primers for Sanger sequencing, PCR setup and clean-up kits, sequencing kits, and standards.

Amazing RNAi

RNAi is a specific, potent, and highly successful approach for loss-of-function studies in virtually all eukaryotic organisms. At Thermo Fisher Scientific, we have developed two types of small RNA molecules that function in RNAi: short interfering RNA (siRNA) molecules and microRNAs (miRNA). Thermo Fisher Scientific offers products for RNAi analysis in vitro and in vivo, including libraries for high-throughput applications. Your choice of tool depends on your model system, the length of time you require knockdown, and other experimental parameters.

Effectively knock down gene expression using our proprietary Silencer® Select, Stealth RNAi™, and Silencer siRNAs, Analyze miRNA function with mirVana™ miRNA mimics and inhibitors
Find a complete solution for successful in vivo RNAi experiments including the highest nuclease stability Ambion

Five Simple Tips to Future Proof Your Loan

In the event that you are in the marketplace of mortgage, we’ve got some handy tips for you to make sure your preparation is as beneficial and stable as possible.

Tip#1. Assess the mortgage loan and the current
Tip#2. Refinance mortgage loan to make sure it suits your requirements
Tip#3. Try some management methods and techniques
Tip#4. Check and control against the unexpected financial shortfalls
Tip#5. Prepare for known and unknown factors

Tips To Not Hiring Bad Bankruptcy Attorneys

If you are facing the situation of hiring bankruptcy attorney and you don’t know how to have the right attorney for your particular case, then here are 5 tips to avoid hiring bad attorney.

– Tip #1: Do not wait – just take action!
– Tip #2: Ask around – people around you!
– Tip #3: Go to the court!
– Tip #4: Schedule the consultation!
– Tip #5: Don’t confuse quality with price!