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We narrowed to 849 results for: Met

Showing: 161 - 180 of 849 results

  1. Multiplexed Capture of Promoter-enhancer 3D Chromatin Structures Using CRISPR

    Type
    Blog Post
    ...original CAPTURE method. Both CAPTURE methods outperformed other 3D chromatin methods such as ChIA-PET...capture (3C) based methods, such as 5C or Hi-C (Han et al., 2018). Think of 3C methods as taking a snapshot... in situ of Regulatory Elements) method. The original CAPTURE method was published in 2017 and addressed...Regulatory Elements: The original CAPTURE method The original CAPTURE method requires the creation of a stable...by BirA. From here, the CAPTURE method is similar to other 3C methods: crosslinking captures chromatin...factors, can increase a gene’s transcription. Sometimes enhancers are located thousands of base pairs ...data. While “pictures” generated with current 3C methods provide useful information about chromatin interactions...

  2. Plasmids 101: Common Lab E. coli Strains

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    Blog Post
    ... mk+) Unmethylated DNA not degraded, cell still can methylate DNA   hsdS(rk-,mk-) Unmethylated DNA not...Consequence dam DNA adenine methylase mutation (GATC) Preparing unmethylated DNA, important when trying... XbaI) dcm DNA cytosine methylase mutation (CCWGG) Preparing unmethylated DNA, important when trying...pathway that cleaves methylated cytosine DNA Allows for uptake of foreign (methylated) DNA mrr, Δ(mcrC-mrr...that cleaves methylated adenine or cytosine DNA Allows for uptake of foreign (methylated) DNA recA, ...thiamine metabolism Requires exogenous thiamine for growth ara disruption of arabinose metabolism pathway...plasmids that should not be Dam or Dcm methylated, allows for methylation sensitive restriction enzymes to ...

  3. Save Time with Transient Plant Leaf Transformations

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    Blog Post
    ...Unver and Budak, 2009). This method works well in C. roseus. A common method for overexpressing genes In... tissue (see Figure 3). This method is probably the most scalable method. Transfer your plant into the..., and Hikmet Budak. "Virus-induced gene silencing, a post transcriptional gene silencing method." International...roseus is strongly limited by the availability of methods. Infection with Agrobacterium rhizogenes can be...stable hairy root lines in tissue culture. This method is commonly used in C. roseus, but processes specific...transgenic cultures or plants, our transient expression method enables us to quickly screen and investigate transcription...problem in non-model organisms. Therefore, transient methods are usually the first choice for investigating ...

  4. RNA Extraction Without A Kit

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    Blog Post
    ...extraction methods evolved into a simple protocol still used today There are many alternative methods for isolating...purification. There is one simple method that works, and variations to that method. A major hurdle to developing...a kit that outlined several reasons why doing something without a kit has advantages: less plastic waste...RNases are commonly found in cells, and without something to block RNase activity upon cell lysis, RNA is... strong protein denaturant would be required- something that broke down RNases before RNases had a chance...Although very effective at isolating total RNA, this method requires a lot of time and depending on how many...guanidinium thiocyanate-phenol-chloroform. In this method, samples are still homogenized and lysed in a guanidinium...

  5. Antibodies 101: Flow Compensation

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    Blog Post
    ...and your cytometer’s laser configuration. Your flow core or an online manual for your cytometer should ...Introduction to Gating in Flow Cytometry Antibodies 101: Flow Cytometry Antibodies 101: Conventional vs...In flow cytometry, compensation is the process of correcting spillover from one fluorescent channel to... chart to find your antibody's spectra). Your cytometer will therefore record the fluorescence for both...called "spillover." For example, if your flow cytometer reads out the fluorophores FITC and PE on the ...at a similar wavelength. However, if your flow cytometer reads them out on different lasers, you should...there are exceptions). For example, on most flow cytometers you will need compensation between APC and APC-Cy7...

  6. Antibodies 101: Secondary Antibodies

    Type
    Blog Post
    ..., it is sometimes assumed that the direct method is more quantitative than the indirect method. But in...indirect approach, while flow cytometry’s standard approach is the direct method. ELISAs, immunohistochemistry... using either the direct or the indirect method.  New Methods Time is a precious commodity in the lab ...signaling molecule has a weak signal. For flow cytometry, secondary antibodies may be indicated on occasion...much easier said than done! Each technique, and sometimes each individual assay, will need to be developed...incubation and wash steps, which means that indirect methods are often significantly longer than direct ones...considered one of the major drawbacks of the indirect method, lengthening an often already lengthy process. ...

  7. Light Sheet Fluorescence Microscopy

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    Blog Post
    ...2015). Nature Methods, 12, 23–26. Pubmed. M. Weber, M. Mickoleit, J. Huisken. (2014) Methods Cell Biol.123... fluorescence microscopy (LSFM). The underlying method was developed in 1902 by Richard Zsigmondy and ...resolution for studying colloidal gold (1).  The method was based on using a thin plane (sheet) of light...sunlight to observe single gold particles with diameters less than 4nm. Lateral view of a cleared rat pons...travel through the same objective lens. Using this method, these authors were able to reconstruct a cleared...slow and unwieldy for large specimens that are millimeters in thickness (8,9).  In addition, there is a ... couple-charged device (EMCCD) or complementary metal-oxide semiconductor (CMOS) sensors, it takes only...

  8. RNA Interference in Plant Biology: New Tools for an Old Favorite

    Type
    Blog Post
    ...from an easy method to identify and quantify actively silencing plants. Non-destructive methods, most popularly...functional studies in unprecedented ways. With the meteoric rise and diversity of CRISPR technologies, one... essential genes. Historically, gene silencing methods were thought to be afflicted by pervasive off-target...discrepancies between gene silencing and knockout methods of the same gene. However, recent work has called...either technology was equivalently precise, but each method recovered different genes known to be required ...Only upon combining the data generated from both methods did a more comprehensive gene list emerge that ... highlights the importance of using orthogonal methods to confidently conclude the function of a gene ...

  9. To Each HIS Own

    Type
    Blog Post
    ...tag to be accessible to the metal ions once the protein is folded. Sometimes a tag placed on one end will...purification methods where binding relies on a specific protein conformation, the polyhistidine-metal ion interaction...sequence, or tag, on the protein. While a variety of methods can be used to successfully isolate proteins, this...affinity of the imidazole ring of histidine for metal ions such as Ni2+ or Co2+ (Hochuli 1988), and can...Hochuli 1988). In this purification approach, the metal ions are immobilized on a bead or resin and a sample...flowed through. The protein of interest binds to the metal ions, becoming immobilized itself. Next, a series...interaction between the protein of interest and the metal ions.      Fig. 1: A sample containing a...

  10. DNA Purification Without a Kit

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    Blog Post
    ...run out of columns. Kit-less DNA purification methods avoid many of the drawbacks of kits and typically...article, we will go through the basics of four methods for DNA purification without a kit as well as one...The plasmid purification and DNA gel extraction method described in Kim and Morrison is nearly identical...filters. Image: Kim and Morrison, 2009. This method provides DNA yield and quality similar to that ...1.5 hours to complete, while this syringe-based method can be completed in 20 to 30 minutes. Additionally...for different purposes, with the Kim and Morrison method, you can just adjust the number of glass filters...working with. Overall, the glass syringe filter method is not only cheaper, but also faster and more flexible...

  11. CRISPR Between the Genes: How to Experiment with Enhancers and Epigenomics

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    Blog Post
    ... region that has mono-methylated and acetylated histones, but not tri-methylated histones, functions as...denote enhancers as follows: (mono_methylation + acetylation) - tri_methylation In the next section we’ll ...H3 me1, me3, and ac – denoting mono-methylation, tri-methylation, and aceylation, respectively (these...of the string, instead of being 10 meters apart, are mere millimeters apart. Similarly, cells compact DNA...coordinates alone aren’t very useful. We’ll need something like Google Maps to help us identify and visualize...field. We’re going to focus on a tried and true method of finding enhancers by isolating genomic regions...proteins,” ChIP-seq from Table 1. Because of DNA’s 3D geometry and the chemical properties of modified histones...

  12. Plasmids 101: Sequence and Ligation Independent Cloning (SLIC)

    Type
    Blog Post
    ...Elledge SJ. Nat Methods. 2007 Mar;4(3):251-6. Epub 2007 Feb 11. PubMed. SLIC: a method for sequence- and...If cloning methods had personalities, SLIC (sequence- and ligation-independent cloning) would be a true...standardized, and rapid multi-part DNA assembly method. Read on to learn how to use it in your research...after transformation. LIC is a reliable cloning method, but it is limited by its sequence constraints....courtesy of Addgene depositor Stephen Elledge. His new method, named sequence- and ligation-independent cloning...although higher DNA concentrations are needed for this method to be successful. Low DNA concentration? No problem...right branch). The mixed PCR and incomplete PCR methods yield a minority of usable product with two 5′ ...

  13. Plasmids 101: Golden Gate Cloning

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    Blog Post
    ...variety of restriction enzyme-based cloning methods. Each method has its own pluses and minuses, but Golden...few advantages over other cloning methods. Exonuclease-based methods like Gibson assembly require 20-40...Golden Gate cloning is one of the easiest cloning methods in terms of hands-on time, as digestion and ligation...sequence homology cannot be assembled using this method, but can be assembled with Golden Gate. The popular...also less expensive than many commercial cloning methods. Golden Gate and synthetic biology Synthetic biologists...Gate cloning into a modular cloning strategy. Sometimes referred to as MoClo, this strategy uses the Type...fragments at a time. As with all Golden Gate-based methods, this system exploits the ability of Type IIS enzymes...

  14. Plasmids 101: Biotinylation

    Type
    Blog Post
    ...substrate Flow cytometry/fluorescence-activated cell sorting (FACS) In situ hybridization Methods for biotinylating...Photobiotinylation is sometimes easier and less expensive than other enzymatic methods. Scientists have commonly...covalently attached to the active site of key metabolic enzymes including biotin carboxylase and decarboxylases... organic acids to produce important cellular metabolites. Biotin acts as a carboxyl carrier from one compound...such as gluconeogenesis, lipogenesis, amino acid metabolism and energy transduction. Biotinylation is the...diet was being supplemented with biotin. Thus something in the egg white was sequestering biotin from ...biotinylated. Scientists have developed several methods to chemically biotinylate macromolecules of interest...

  15. Using Phosphoserine to Study Protein Phosphorylation

    Type
    Blog Post
    ...protein? Classical methods for studying protein phosphorylation A number of classic methods exist for this...MEK1 phosphomimetic residues in the above study with phosphoserine (to create a pSer/phosphomimetic dual... model systems coupled with advances in mass spectrometry suggest that phosphorylation is far more prevalent... most common of which is the utilization of phosphomimetic residues. The phosphorylated residue of interest...revealed a curious anomaly amongst a subset of methanogenic archea: these organisms lacked a classic cysteinyl...but can be made constitutively active through phosphomimetic mutation of these residues. Due to the limitations...limitations previously mentioned, phosphomimetic mutations on the activation loop do not fully activate ...

  16. Recombinase-based State Machines Enable Order-dependent Logic in vivo

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    Blog Post
    ... within a sample using a metric that we describe in the Materials and Methods section of our paper (1)...expensive, then the qPCR method should be considered as an alternative. The qPCR method can be performed on...perturbation. Also, this method requires live, intact cells; the sequencing and qPCR methods do not. How might...fluorescent proteins in each state. The three methods of state interrogation mentioned above (Sanger ...Sanger sequencing is simple and the most accurate method as it directly reads the state of a register. However... of registers in a sample based on a similarity metric that we defined in our paper. In our opinion, the...convenient strategy is the fluorescent reporter based method since it lends itself to cheap, quick, and high...

  17. Hot Plasmids: FRET-Based Biosensors

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    Blog Post
    ...shy away from ratiometric imaging, he recommends they give it a shot. “Beginners sometimes think it’s complicated...imaging of transition metal ions, such as zinc, copper, and iron. “These are essential metal ions, but they ...neurons and T lymphocytes. Nat Methods. 2014 Jan 5. doi: 10.1038/nmeth.2773. (PubMed) Friedrich et al....which are available at Addgene, can be used for ratiometric in vivo imaging. Griesbeck explains, while these...possible for added sensitivity. As reported in Nature Methods, the FRET changes of these Twitch sensor variants...both neuroscience and immunology. Don’t be shy: Ratiometric imaging for beginners Griesbeck says his calcium...important and when? That is not known.” Transition Metals Maarten Merkx of Technische Universiteit Eindhoven...

  18. Sequencing Options for CRISPR Genotyping

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    Blog Post
    ...older assays. Biased sequencing methods There are two primary methods of off-target detection: biased ...off-target effects of sgRNAs using computational methods. They identify possible off-target sites across...that they can later sequence for mutations. One method a researcher can use to test predicted off-target...disastrous health effects in the clinic. NGS-based methods provide the most complete information profile regarding...editing events. Two of the most common cell-based methods are genome-wide, unbiased identification of double-strand...all cell types Must be verified with cell-based method IDLV 1% Cell-based Programmable, can detect DSBs...in live cells Not as senstive as other unbiased methods, high background BLESS Not reported Cell-based...

  19. Quick Guide to Near-Infrared Fluorescent Proteins

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    Blog Post
    ...eukaryotic cells as an intermediate of heme metabolism (Shemetov et al, 2017). Modern state-of-the-art NIR...iGECI calcium indicator (Shemetov et al., 2021). In addition to FRET, intensiometric calcium indicators NIR-GECOs... in NIR. Usually, mercury lamps are suboptimal, metal halide lamps are better, and xenon lamps are the...daria_gift_shch.     References and resources References Shemetov, A.A., Oliinyk, O.S. & Verkhusha, V.V. How to ...near-infrared fluorescent calcium ion indicator. Nat Methods 16, 171-174 (2019). Matlashov, M.E. et al. A set...scales. Nat Commun 11, 239 (2020). Oliinyk, O.S., Shemetov, A.A., Pletnev, S., Shcherbakova, D.M. & Verkhusha... antigen-dependent fluorescent nanobodies. Nat Methods submitted (2021). Tchekanda, E., Sivanesan, D. ...

  20. Hassle-free 96-well Format Epitope Tagging Using Cas9 Ribonucleoprotein

    Type
    Blog Post
    ...Nucleofection method for delivery of csRNP reagents into cells. Optimization of delivery methods (electroporation...-IN” design tool with the csRNP method. We anticipate that our method will open up immense possibilities...pull-down assays include immunoprecipitation mass spectrometry (IP/MS) and chromatin immunoprecipitation (ChIP... protein complexes are then analysed by mass spectrometry to identify the interacting proteins. Similarly...of the target protein. Both IP/MS and ChIP-seq methods provide a wealth of information about how protein...antibody (Jarvik and Telmer 1998). Indeed, this method can be used at high throughput because numerous...Bressan et al. 2017; Liang et al. 2017). Current methods of epitope tagging, however, suffer from many pitfalls...
Showing: 161 - 180 of 849 results