... editing is a “search and replace” gene editing method in which a reverse transcriptase (RT) is fused ...

...elegans and other nematodes. Wormbuilder - Describes methods to engineer the genome of the genetic model organism...

...on factors such as selectable marker or cloning method. When using CRISPR, you will need to express both...

...standards to calibrate the numerous and disparate methods used to measure FRET. Plasmid FRET Pair C5V Cerulean...

...modified and amplified (through standard cloning methods). There are different types of viruses that are...

...non-invasive live imaging. A standard toolbox of genetic methods, such as Tol2 transposons, Cre-mediated site-specific...

...on factors such as selectable marker or cloning method. When using CRISPR, you will need to express both...

... . Insertional chromatin immunoprecipitation: a method for isolating specific genomic regions. Hoshino...

... that responded to stimuli, such as hormones or metal ions; however, these systems were confounded by ...

...efficient guide (most guides would work, but they sometimes have different efficiency). And it is best if ...

...depend on several factors including: the delivery method, mouse strain, target cell type or tissue of interest...

...evaluated in mouse brains, using various delivery methods including intracerebroventricular (ICV) delivery...

...protein of interest resides. There are a variety of methods for determining subcellular localization. One of...

...Overview Quantification Methods Capture Methods Visualization Methods Cell Sorting Methods Multiplex Assays ...Antibody Applications - Cell Sorting Methods Flow cytometry Flow cytometry, used commonly in immunology, allows... through the indirect detection method. In the direct detection method, the signaling molecule is conjugated...Antibody Applications - Quantification Methods Quantification methods, such as western blots or ELISAs, are...output measured. This method can be performed using the indirect detection method via a conjugated secondary...sample. Controls for quantification methods Quantification methods should be run with: a positive control...included. Antibody Applications - Capture Methods Capture methods such as immunoprecipitation (IP) and chromatin...

...toxins, cell viability, and tumor metastasis. One of the most popular methods for conducting genome-wide screens...activation through cytosine demethylation DNMT3A - repression through cytosine methylation MQ1 - repression through... to conventional methods for genomic purification, CRISPR-based purification methods are more straightforward... currently no defined measure of fidelity, so sometimes they will be referred to as increased fidelity...Cas9’s proofreading capabilities. No matter the method, increased fidelity enzymes generate less off-target... editing is a “search and replace” gene editing method in which Moloney Murine Leukemia Virus Reverse ...Although lentiviral libraries are the most popular method for CRISPR screening, they are not suitable for...

...fused a TALEN to the Tet1 demethylase catalytic domain, could mediate demethylation and induce transcription... fused a TAL effector to LSD1 histone demethylase to demethylate enhancer regions (Mendenhall et al., ...Tet1 demethylase Ronggui Hu’s lab has created pdCas9-Tet1-CD for targeted cytosine demethylation in mammalian... dCas9-Tet1 demethylates cytosines at promoters and enhancers. This targeted demethylation is associated... dCas9-DNMT3A methylates cytosines at promoters and enhancers. This targeted methylation is associated... Q147L mutation improves methylation kinetics such that cytosine methylation occurs within 24 hours rather...CRISPR-based DNA demethylation by Casilio-ME-mediated RNA-guided coupling of methylcytosine oxidation and...

...in their DNA methylation patterns, either large increases or decreases. Hypermethylation of CpG islands...hybridization, methylation-sensitive restriction enzyme digestion, DNA adenine methyltransferase identification...linker is a methyllysine binding domain (chromodomain), which binds selectively to lysine-methylated peptides...the results. When methylation occurs at the histone-derived peptide, the methyllysine binding domain causes... (pcDNA3-K9 histone methylation reporter) and K27 (pcDNA3-K27 histone methylation reporter) positions ...to recognise lysine 27 methylation. When tested in living cells where methylation of these sites was controlled...significantly different FRET levels between unmethylated and methylated states. Thus, this system is a reliable...

...efficiency and cross-talk. Methods to Measure FRET for Cell Biology Studies Several methods have been used over...many measurement methods as feasible when first beginning to establish the FRET methodology for a given experiment... this method useful for dynamic experiments in which FRET changes are large. Two other methods are commonly... photobleaching method and the fluorescence lifetime imaging microscopy (FLIM) method. Acceptor photobleaching...photobleaching method The acceptor photobleaching method is simple but limited to a single measurement. This...imaging microscopy method FLIM has been developed more recently and is the most rigorous method for measuring...pitfalls and to use it at its best. What are the Parameters that Affect FRET? FRET occurs when the two fluorophores...

...and chemiluminescent methods that require specialized equipment. Chromogenic methods are not as sensitive... Because the method uses a single antibody, it is less specific than other ELISA methods. Users may also...drawbacks of the competitive ELISA method are similar to those of the direct method. Namely, since the assay requires...advantages and disadvantages of each ELISA method Method Advantages Disadvantages Direct Fast and...enzyme-linked immunosorbent assay (ELISA) is a versatile method used to quantify the level of target antigen in...of each, and some considerations when using the method. ELISA basics All ELISAs follow the same overall... discuss the more common enzyme-based reporter methods. For enzymatic signal detection, users provide ...

...expression. CRISPRa methods vary in their transcriptional activators: some methods rely on fusion proteins...Newer methods for CRISPR activation While SAM, SunTag, and VPR have become the most popular methods for ...transcriptional programming. Nat Methods 12:326–328 . https://doi.org/10.1038/nmeth.3312 Chavez A, Tuttle M, ... in multiple species. Nat Methods 13:563–567 . https://doi.org/10.1038/nmeth.3871 Konermann S, Brigham...transcription factors. Nat Methods 10:973–976 . https://doi.org/10.1038/nmeth.2600 Qi LS, Larson MH, Gilbert... 2013). In the years that followed, innovative methods greatly improved CRISPRa, expanding its practicality...al., 2015, Chavez et al., 2015). Most popular methods for CRISPR activation CRISPR activation uses dCas9...