Improved protocol for recovery of bacterial DNA from water filters: Sonication and backflushing of commercial syringe filters.
Extraction of bacterial DNA from water filters is frequently used in aquatic microbial ecology, e.g., for sequencing-based diversity studies. Here, we describe an improved size-class filtration and bacterial DNA extraction protocol using commercial “syringe filters”, involving sonication- and backflushing-steps and DNA-binding magnetic beads. Importantly, the sonication-step improved DNA recovery up to 10-fold. The DNA extraction-turnaround time is 4h, with a hands-on time of about 2h.
Fragmentation of DNA in a sub-microliter microfluidic sonication device.
Fragmentation of DNA is an essential step for many biological applications including the preparation of next-generation sequencing (NGS) libraries. As sequencing technologies push the limits towards single cell and single molecule resolution, it is of great interest to reduce the scale of this upstream fragmentation step.
Here we describe a miniaturized DNA shearing device capable of processing sub-microliter samples based on acoustic shearing within a microfluidic chip. A strong acoustic field was generated by a Langevin-type piezo transducer and coupled into the microfluidic channel via the flexural lamb wave mode.
Purified genomic DNA, as well as covalently cross-linked chromatin were sheared into various fragment sizes ranging from ∼180 bp to 4 kb. With the use of standard PDMS soft lithography, our approach should facilitate the integration of additional microfluidic modules and ultimately allow miniaturized NGS workflows
Fragmentation of DNA by sonication.
INTRODUCTIONDNA fragmentation is often necessary prior to library construction or subcloning for DNA sequencing. This protocol describes a method for DNA fragmentation by sonication. During sonication, DNA samples are subjected to hydrodynamic shearing by exposure to brief periods of sonication.
DNA that has been sonicated for excessive periods of time is extremely difficult to clone. Most sonicators will not shear DNA to a size of less than 300-500 bp, and it is tempting to continue sonication until the entire DNA population has been reduced in size. However, the yield of subclones is usually greater if sonication is stopped when the fragments of the target DNA first reach a size of ~700 bp.
Description: DNA Cleanup Magnetic Beads are superparamagnetic, non-aggregating iron oxide particles (or ‘microspheres’) which can bind DNA ant high capcity. The highly efficient DNA purification magnetic beads are based clean-up system for the purification of DNA for NGS workflows. Superior yield, purity, and quality over the leading competitors, all at a better value, because we design and manufacture them in-house. DNA Cleanup Magnetic Beads enables faster binding kinetics, with high sensitivity & selectivity, in both manual and automated biomedical and research applications. The purified DNA is of high yield and integrity and is free of inhibitors, ready for use in a number of downstream applications including PCR, qPCR, mutation screening, microarray analysis, sequencing, single nucleotide polymorphism (SNP) and short-tandem repeat (STR) genotyping.
Description: The Magnetic Beads are ideal for DNA purification, concentration and size selection, along with next-generation sequencing library preparation kits.
Description: Anti-HA magnetic beads kit is based on hydroxyl magnetic beads covalently coupling with high quality mouse IgG2b monoclonal antibody, used for co-immunoprecipitation and protein purification.
Description: Anti-HA magnetic beads kit is based on hydroxyl magnetic beads covalently coupling with high quality mouse IgG2b monoclonal antibody, used for co-immunoprecipitation and protein purification.
Description: Anti-Myc magnetic beads kit is based on hydroxyl magnetic beads covalently coupling with mouse IgG1 monoclonal antibody.it is recommended to use for co-immunoprecipitation and protein purification.
Description: Anti-Myc magnetic beads kit is based on hydroxyl magnetic beads covalently coupling with mouse IgG1 monoclonal antibody.it is recommended to use for co-immunoprecipitation and protein purification.
Description: Anti-Flag magnetic beads is based on hydroxyl magnetic beads covalently coupling with mouse IgG1 monoclonal antibody. It is recommended to use for co-immunoprecipitation and protein purification.
Description: Anti-Flag magnetic beads is based on hydroxyl magnetic beads covalently coupling with mouse IgG1 monoclonal antibody. It is recommended to use for co-immunoprecipitation and protein purification.
Description: The Streptavidin-Magnetic Beads are 2.8 µm superparamagnetic particles covalently coupled to a highly pure form of streptavidin (SA). The beads can be used to capture the biotinylated proteins or other molecules, because Streptavidin (SA) has an extraordinarily high affinity for biotin with a dissociation constant (Kd) on the order of 10−14 mol/L, the Biotinylated molecules can bind to the SA irreversibly.Streptavidin is a tetrameric protein purified from the bacterium Streptomyces avidin, and exhibits high binding affinity for biotin. Able to bind one molecule of biotin with each subunit. Streptavidin (PI=6.0-7.5) has lower level of non-specific binding to various biological components at physiological pH than avidin (PI=7.4), resulting from its isoelectric point (PI).The Streptavidin-Magnetic Beads is easy to capture the biotinylated proteins or other molecules in Chemiluminescence procedures, and the bounded protein have no activity lost, this ready to use products could greatly save your protein coupling time and hassle, and help us get the best performance and highly reproducible results.
Description: The biotinylated FAP protein was conjugated to streptavidin magnetic beads. This pre-coupled magnetic bead product can capture the anti-FAP antibody from various assay systems. The beads are in uniform size, narrow size distribution with large surface area and unique surface coating, which can help you get the best performance and highly reproducible results. This FAP coupled magnetic beads will bring great convenience with minimum non-specific binding and developed protocols. This ready-to-use product could greatly save your time and hassle.
Description: Human CD19 protein is expressed from human 293 cells (HEK293). It contains AA Pro 20 - Lys 291(Accession # P15391-1).
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Chromatin immunoprecipitation (ChIP): revisiting the efficacy of sample preparation, sonication, quantification of sheared DNA, and analysis via PCR.
The “quantitative” ChIP, a tool commonly used to study protein-DNA interactions in cells and tissue, is a difficult assay often plagued with technical error. We present, herein, the process required to merge multiple protocols into a quick, reliable and easy method and an approach to accurately quantify ChIP DNA prior to performing PCR.
We demonstrate that high intensity sonication for at least 30 min is required for full cellular disruption and maximum DNA recovery because ChIP lysis buffers fail to lyse formaldehyde-fixed cells. In addition, extracting ChIP DNA with chelex-100 yields samples that are too dilute for evaluation of shearing efficiency or quantification via nanospectrophotometry.
However, DNA extracted from the Mock-ChIP supernatant via the phenol-chloroform-isoamyl alcohol (PCIA)method can be used to evaluate DNA shearing efficiency and used as the standard in a fluorescence-based microplate assay.
This enabled accurate quantification of DNA in chelex-extracted ChIP samples and normalization to total DNA concentration prior to performing real-time PCR (rtPCR). Thus, a quick ChIP assay that can be completed in nine bench hours over two days has been validated along with a rapid, accurate and repeatable way to quantify ChIP DNA.
The resulting rtPCR data more accurately depicts treatment effects on protein-DNA interactions of interest.
Rapid quantification methods for genetically modified maize contents using genomic DNAs pretreated by sonication and restriction endonuclease digestion for a capillary-type real-time PCR system with a plasmid reference standard.
For rough quantitative analysis of genetically modified maize contents, rapid methods for measurement of the copy numbers of the cauliflower mosaic virus 35S promoter region (P35S) and MON810 construct-specific gene (MON810) using a combination of a capillary-type real-time PCR system with a plasmid DNA were established.
To reduce the characteristic differences between the plasmid DNA and genomic DNA, we showed that pretreatment of the extracted genomic DNA by a combination of sonication and restriction endonuclease digestion before measurement is effective.
The accuracy and reproducibility of this method for MON810 content (%) at a level of 5.0% MON810 mixed samples were within a range from 4.26 to 5.11% in the P35S copy number quantification. These methods should prove to be a useful tool to roughly quantify GM maize content.
