...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′ ...

...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...

...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...

...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 ...

... 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...

...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...

...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...

...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. ...

...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...

...site for inserting a custom gRNA.  While this methodology has proven to be incredibly valuable to scientists...complications that must be considered when using this method:     Cells must be amenable to transfection or... to using RNPs for CRISPR experiments. The RNP method can often be used in cells that are difficult to...CRISPR plasmids) are not expressed. Because this method does not require the delivery of foreign DNA, and...CRISPR experiments using RNPs is the variety of methods that can be used to deliver a Cas9-gRNA RNP. This...systems that may not be amenable to plasmid based methods. One of the most common techniques for delivery...demonstrated the use of cationic lipid-mediated methods to deliver Cas9-gRNA RNPs to hair cells in the ...

...sensitive, high-throughput method to accelerate characterization of nucleic acid metabolic enzymes." Nucleic acids... nicks with no gaps. For example, DNA assembly methods, such as Gibson Assembly® and NEBuilder® HiFi DNA... use fluorophore- quencher pairs or qPCR-based methods to greatly increase the sensitivity of detection...Biolabs® (NEB) researchers recently published a method for the high-throughput profiling of ligase fidelity...high-sensitivity multiplexed format (21, 27). This methodology has been used to rapidly screen buffer conditions...DNA Ligase and their effect on fidelity. In this method, substrate pools were prepared consisting of one...capillary electrophoresis (CE) (see below). This method allowed screening of all possible base combinations...

... hybridization method (DNA dot blotting) had been used to titrate AAV, but this method is not widely used...2012). PCR-based methods are robust, easy, fast, and convenient. However, these methods are not necessarily...vectors and improved alternative methods." Human Gene Therapy, Part B: Methods 23.1 (2011): 1-7. PubMed PMID...of a viral vector solution, and generally these methods measure different characteristics of the solution...viral genomes (by qPCR or other DNA quantification methods - see below), since each viral particle typically...viral vector preparations. Addgene has used this method to generally validate our viral vector preparation... typically calculated by two popular PCR-based methods: Quantitative PCR (qPCR) Digital droplet PCR...

...Journal of Neuroscience Methods, 172(2), 250–254. https://doi.org/10.1016/j.jneumeth.2008.05.00...make when designing the experiment. What detection method? What membrane? What should you block with? It...time to two days.  Indirect or direct detection method Indirect detection is the most common way to run...source, making it easy to switch from one readout method to another or change primary antibodies as needed...proteins, instead of the more common chemiluminescent methods for detection (though the enzymes used for chemiluminescence...denaturing blot.  There are a number of lysing methods and buffers to choose from. If you are starting... tissue, you’ll likely want to use a mechanical method before your enzyme lysis. For cell cultures, enzyme...

...you can detect it through the APC-Cy7 parameter in the cytometer's software. It also means we should exclude...Introduction to Gating in Flow Cytometry Antibodies 101: Flow Cytometry Antibodies 101: Flow Compensation...When analyzing your cells using flow cytometry, you are typically measuring the presence or absence of... to give you the appropriate readout. A common method of labeling proteins is to fuse them with a fluorescent...reporters, and/or dye stains used in your flow cytometry experiment are known as your "panel". In this ...strength of the emitted photons detected by the cytometer. Brighter fluorophores have a stronger signal....from: https://emea.bd.com/panel-design/en/flow-cytometry-fluochrome-brightness-spillover Excitation...

...study protein-protein interactions. Flow cytometry Flow cytometry is used to detect proteins on the surface...expression: The nuts and bolts. Methods 116:51–62 . https://doi.org/10.1016/j.ymeth.2017.01.009 Crivianu-Gaita...immune system production and many more engineered methods. Each type of antibody and production strategy ...recognizes the primary antibody and provides the method of detection. You’ll also want to use the appropriate...Enzyme-linked Immunosorbent Assay). Immunoprecipitation Sometimes, instead of just detecting the presence of protein...the mixture. Antibodies can do this, too! In this method, antibodies bound to microscopic beads grab the...The fluorescence signal is detected by the flow cytometer and functions as a proxy for protein expression...

...habitat (Ronda et al., 2019). They called their method Metagenomic Alteration of Gut microbiome by In situ Conjugation...Carlotta, et al. "Metagenomic engineering of the mammalian gut microbiome in situ." Nature methods 16.2 (2019... microbes. Let’s take a look at a few of these methods below. Engineering gut microbes in situ While...Harris Wang lab at Columbia University developed a method for genetically altering gut microbiome members... and diagnostic labs, deep sequencing and qPCR methods are too costly and time intensive to process a ...microbiome, James Collins’s lab developed a paper-based method that can be used to detect microbes and biomarkers...biomarkers on paper. At the heart of their method is the RNA toehold switch, a RNA hairpin that can be designed...

...drawbacks of different methods (for comprehensive protocols for all of the methods discussed here refer ... in a matter of hours. A major drawback of this method, however, is that a p24 ELISA measures all p24 ... direct p24 measurement. Like a p24 ELISA, this method is relatively quick, providing results in less ... on the vector backbone. When using qPCR based methods for viral titration consider using a reverse transcriptase... Functional titer is considered a more accurate method to quantify virus as it only measures infectious...infectious viral particles. One of the si mplest methods of measuring functional titer uses FACS to count the...transduction with serial dilutions of a viral prep. This method is particularly useful when vectors carrying fluorescent...

...acetone, methanol, or ethanol, work by dehydrating and precipitating cellular components. This method of fixation...introduction to immunofluorescence (IF) - a common method for visualizing molecules of interest within a ...tissue. In that introduction, we broke down the method into six general steps and outlined the considerations...saturated being 37%). Formalin solutions often contain methanol as well, in order to slow the polymerization of...recover good antibody binding by tweaking those parameters, but in some cases your antibody may just not...experiment.   Figure 1: Choice of fixation method impacts antibody binding. Formaldehyde cross-links...hidden within the folded protein (green region). Methanol dehydrates the protein, which can disrupt protein...

...then eluted. This method is appropriate for most situations.  A slightly different method is desirable if...typically carried out in one of two ways. Both methods utilize the same basic strategy: immobilize the...target protein is eluted.     In the first method, an antibody against a target protein is immobilized...if the target is present in low amounts. In this method, the free antibody is incubated with the protein...antibody-protein complexes, and as in the first method, unbound proteins are washed away and the target...protein. One of the most common immobilization methods utilizes bacterial cell wall proteins, Protein ..., the target protein can be eluted. The elution method will vary depending on the specific target protein...

...Legionella pneumophila, Methylophilus methyltrophus, Methylococcus methanolicus, Methylosinus trichosporium, Myxococcus...Klebsiella aerogenes, K. pneumoniae, Methylophilus methylotrophus, Moraxella spp., Mycobacterium aurum...Klebsiella spp., Legionella pneumophila, Methylophilus methylotrophus, Myxococcus virescens, M. xanthus, Proteus...plasmids may have high copy numbers and reduced metabolic load (Meyer 2009). Figure 1: Idealized ...eutrophus, Azotobacter vinelandii, Brevibacterium methylicum, Caulobacter crescentus, Desulfovibrio vulgaris...plasmid vectors for gene expression in bacteria. Methods in Molecular Biology (Clifton, N.J.), 765, 327–...