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... differences between transformation, transduction, conjugation, and transfection Resources on Addgene.org... in 3 different ways: Transformation grade DNA Cloning grade DNA Transfection grade DNA Figure...Figure 1: Transformation introduces transformation grade plasmids into a bacterial cell. Transformation...plasmids into mammalian cells? Use transfection grade DNA. Transfection is the process of inserting an ...kits provide extra washing steps to favor the removal of this agent to increase transfection success.  In...Transformation grade DNA  Transformation is the process of inserting exogenous DNA in a microorganism. The amount ...plasmid DNA that is necessary for bacterial transformation of chemically and electrocompetent cells is...

...validated using at least one of these strategies. Choosing which strategy to use will depend on your intended... (1) other strategies may not be feasible and (2) large-scale proteomic and transcriptomic datasets already... Recombinant Strategies This approach is essentially the opposite of the Genetic strategy and looks for...Interest (GOI), and transfect it into your favorite cell line. In parallel, you transfect cells with another...(Independent) Antibodies Strategies These strategies use multiple, unique antibodies against the same ... antibody has arrived, hooray! Before you jump straight into the exciting new experiments that you’ve ...may need to roll up your sleeves and perform some extra validation experiments yourself.  Goal of Antibody...

...copy number found in plants makes traditional knockouts unattractive from a practical perspective. Therefore...non-exclusive gene silencing pathways: transcriptional and post-transcriptional gene silencing (PTGS). In either...silence the target gene(s) either transcriptionally or post-transcriptionally. In plants, different dicer-like...where approximately 20-30 nucleotides of double-stranded RNA (dsRNA) results in downregulation, or “silencing...either pathway, short double-stranded RNA fragments (siRNAs) are produced from long dsRNA via an RNase III... through targeted mRNA degradation, blocking translation, or chromatin modifications of complementary ... Figure 1: Schematic of an RNAi-inducing transgene. Typically, the desired target of RNAi is expressed...

...the Roska lab used four different design strategies: Strategy #1 (ProA): Create promoters with sequences...specificity.  Strategy #3 (ProC): Create promoters by repeating mouse cell type specific transcription factor...were transcriptionally active in retinal cell types in mice. Figure 1: The four strategies used...synthetic promoters. Legend: TSS: transcription start site. TFBS: transcription factor binding site. Image from... sparse labeling of neurons for tracing experiments.  Neuronal tracing experiments: While synthetic promoters...for neuronal tracing experiments where lower levels of labeling makes it easier to trace a neuron’s projections...particular cell type is important not only for translational research, such as gene therapy, but also basic...

... in vivo administration, which results in the death of transduced cells and loss of transgene expression...produced at high titers, can accommodate large transgenes, transduce quiescent and dividing cells, and do not...recombinant pAdV plasmid is identified, re-transform into a bacterial strain not prone to recombination for amplification... Keith L. March, and Bruce C. Trapnell. "Evaluation of the concentration and bioactivity of adenovirus...Adenoviral vectors (AdV) are attractive vectors for research applications and gene therapy: they can ...adenoviral genome. The genome itself is a linear double-stranded DNA of ~36kb, with inverted terminal repeats (...E3 regions and can accommodate up to 8.2 kb of transgene DNA. The second generation vectors are deficient...

...are fused to two separate proteins. (In contrast, intramolecular FRET probes contain the donor and acceptor...this FRET you speak of? Förster Resonance Energy Transfer (FRET) was originally described by Theodor Förster...alternative acronym Fluorescence Resonance Energy Transfer. Unlike the typical excitation and emission of...excited fluorophore, FRET involves a non-radiative transfer of energy (i.e. no emitted photons) from the excited...excitation by absorption of a photon 2. Energy transfer from the excited donor directly to the acceptor... acceptor In order for FRET to occur several constraints must be met. The donor and acceptor fluorophores...the acceptor fluorophore. Otherwise the energy transferred from the donor will not be able to excite the...

... interest.  SapTrap Tool Kit for CRISPR Cas9 edits At Addgene, we have the entire SapTrap toolkit from...17986455. PubMed Central PMCID: PMC2238901. Schwartz, Matthew L., and Erik M. Jorgensen. "SapTrap, a toolkit...26837755. PubMed Central PMCID: PMC4905529.  Schwartz, M. L., & Jorgensen, E. M. "SapTrap vectors for introducing... PubMed Central PMCID: PMC5693259.  Wang, Han, et al. "Split cGAL, an intersectional strategy using a ...PubMed Central PMCID: PMC4689222. Additional resources on the Addgene blog Learn more about SapTrap Read...scientists have been continuously sharing their worm strains through the Caenorhabditis Genetics Center (CGC...CGC) which maintains and distributes the strains all over the world. When I was at the 22nd International...

...light to regulate transcription, also includes light-regulated histone methyltransferases and deacetylases...CRISPR Epigenetics resource. Transcriptional activation p300 acetyltransferase dCas9 fused to the catalytic...other Tet1 delivery systems. Transcriptional repression DNA Methyltransferase 3 Alpha (DNMT3A) Vlatka Zoldoš...associated with transcriptional repression. Created with BioRender.com. DNA Methyltransferase MQ1 Margaret...Fusing dCas9 to a transcriptional activator like VP64 or VPR activates transcription, whereas dCas9-KRAB...domain, could mediate demethylation and induce transcription at CpG regions of various promoters. Other researchers...modifiers to specific loci without inducing double strand breaks. Below you'll find some of the CRISPR-based...

...which cleaves one strand of DNA upon binding the protospacer. The resulting double strand break (DSB) stimulates...target site, but does not cut either strand of DNA. Traditional Cas9 regulators In addition to its ...two catalytic domains, HNH and RuvC, cleave DNA strands complementary and non-complementary to the gRNA... then be fused to effector domains such as transcriptional activators or repressors. These fusion proteins...loci within a single cell. One might imagine transfecting cells with both the nuclease-positive and nuclease-null...target site (1). Although this is possible, this strategy suffers from several limitations. Perhaps most...Consequently, fewer genetic tools for targeted transcriptional and epigenetic regulation have been validated...

...Dawson lab Re-transformation of the SPLINTR Libraries Protocol.   Selecting your strain for amplification...ensure the highest possible transformation efficiency. Using Stbl4 or other ultra-high efficiency cells ensures...libraries, chemical transformations can also be used. Efficiency for chemical transformations is less than that...for your transformation(s). The amount of competent cells you need total and per transformation may vary...the manufacturer’s protocol for transformation and expected transformation efficiency, or more specifically...with a large backbone might be more difficult to transform.    Figure 1: Factors to consider when ... coverage, since this will help decide what transformation efficiency you need to achieve. Most pooled...

...Chemical Genomics and Translational Medicine 3.1 (2009). PubMed PMID: 20161833. PubMed Central PMCID: PMC2802762...23667515. PubMed Central PMCID: PMC3647052. 11. Stryer, Lubert. "Fluorescence energy transfer as a spectroscopic...is it? What parts of it move when you feed it substrate or add an essential cofactor? How many binding...what's it good for? Fluorescence resonance energy transfer (FRET) is a biophysical interaction between two...first fluorophore (the donor) is excited, it can transfer energy directly via resonance to the second fluorophore...LanthaScreen) (Degorce et al., 2009), measuring intracellular calcium (Miyawaki, Griesbeck, Heim, & Tsien,...structural changes in the ribosome during protein translation (Ermolenko et al., 2007). All that is required...

...genetically tractable. Mice can be easily manipulated with tools like CRISPR to make transgenic lines. The...28804628. PubMed Central PMCID: PMC5525645. Mello, Craig C., et al. "Efficient gene transfer in C. elegans...elegans: extrachromosomal maintenance and integration of transforming sequences." The EMBO journal 10.12 (1991...externally. Fruit flies are also relatively straightforward to manipulate genetically. Scientists can make... in only 6 weeks compared to months to make a transgenic mouse line. Over the years the fly has become...DNA or RNA to edit their genomes or to create transgenic animals. Zebrafish are an ideal model organisms...development studies as their embryos are completely transparent. Thus scientists can easily observe early developmental...

...to measure stickiness. In vitro, ultracentrifugation or gel filtration is the most common analytical method...random insertions can be generated with a transposon-based strategy (Sheridan, 2002). Avoiding protein over-expression...polarization independent of GDI-mediated extraction and actin-based trafficking." PLoS biology 13.4 (2015): e1002097...physiologic conditions." Traffic 13.5 (2012): 643-649. PubMed PMID: 22289035. PubMed Central PMCID: PMC3324619...vitro transposition reaction." BMC neuroscience 3.1 (2002): 7. PubMed PMID: 12086589. PubMed Central PMCID...engineering of fusion proteins is relatively straightforward. However, generating a fusion while keeping...interest can be challenging. In this blog I discuss strategies to generate fusion proteins and highlight some...

...MSN2 transactivation domain. Transcription factor activation via the appropriate ligand - estradiol for...GAL4/estradiol controlled transcription factor and a ZIF268/progesterone controlled transcription factor...ZIF268. To demonstrate independent control of gene expression, Aranda-Díaz et al transformed yeast with...specific to each transcription factor. They then induced cultures of these transformants with various concentrations... levels. That is, if transcription factor 1 controls expression of transcription factor 2, which directly...pH-based biosensors used to detect the maturation of neutral vesicles to more acidic lysosomes. Though a number...Tyler J. Ford Listen to the Synthetic Yeast Transcription Factors Podcast Segment Researchers from the...

...between the edited strand and wild-type strand of the host DNA. When these strands hybridize, the nuclease...PubMed Central PMCID: PMC4349094. 3. Bell, Charles C., et al. "A high-throughput screening strategy for ... known as CRISPR genotyping, is critical to demonstrating causal relationships between genotype and assayed...nuclease can detect strands with mismatches and cleave them. The results are then visualized using gel electrophoresis...is also key to perform these validations as translational researchers begin to use CRISPR as a therapy...nucleotides) that may still lead to off-target double-stranded breaks (Tsai et al. 2015). Additionally, current...et al. 2015) for cell-based experiments. These strategies can be used in concert with in silico prediction...

...showed that the DNA extract is a heterogeneous mix of single-stranded and double-stranded species. While not...molecules in the DNA extract associate with complementary strands, enough double-stranded species exist in...previously require a preliminary double stranding step. The double stranding step was assumed to be necessary...genome is single-stranded and both TA-based ligation and tagmentation require a double-stranded template.  ... [-] strands that are packaged at the same frequency, we reasoned that, following DNA extraction, the ...the [+] and [-] strands might naturally associate forming a double-stranded species that could be used as... of the main tenets of our quality control (QC) mantra is to do our utmost to ensure that scientists are...

...humanized wt Cas9, dCas9 (transcriptional repressor), and dCas9-VP64 (transcriptional activator)-containing...Multiplexing from a single transcript You can also multiplex gRNAs via a polycistronic transcript. Rather than being...being transcribed from different promoters, the gRNAs are transcribed together and are flanked by specific...simultaneously. Vectors for both transient expression and Agrobacterium-mediated transformation are available. For ...system for processing a continuous multi-gRNA transcript, each gRNA must be expressed from its own promoter.... But that doesn’t mean you have to clone and transfect multiple promoter-gRNA constructs in order to ...destination vector that expresses Cas9. This is illustrated in the schematic below.   Figure 2: ...

... Infectious titer: the concentration of viral particles that can transduce cells. Infectious titers ...PMID: 22428975. PubMed Central PMCID: PMC3640491. 2. Grimm, D., et al. "Titration of AAV-2 particles via...PubMed Central PMCID: PMC5445070. 6. Wang, Feng, et al. "A reliable and feasible qPCR strategy for titrating....qPCR...Digital droplet PCR...DNA dot blot...Transduction assay...SDS-PAGE...Electron microscopy… Any ...can AAV titers tell you? Physical titer: the concentration of viral particles containing viral genomes....Physical titers are measured by quantifying the concentration of viral genomes (by qPCR or other DNA quantification...titer of a viral vector stock can vary based on transduction target and can also be altered by freeze-thaw...

...dimerization.   1. Transmembrane fluorescent protein fusions Transmembrane proteins, i.e. receptors...receptors and transporters, are integrated into lipid bilayers. Fusion of an FP to a transmembrane protein significantly...physiologic conditions. Traffic, 2012. 13(5): p. 643-9. PubMed PMID: 22289035. PubMed Central PMCID: PMC3324619...Green Fluorescent Protein (GFP/EGFP) for a transcriptional reporter or as a general cytoplasmic label ...too common issues follow. It's a matter of concentration Most FPs in nature are prone to dimerization...dimerization (i.e. EGFP) [1, 2] or even form obligate tetramers (i.e. DsRed) [3]. This is a problem for fusion proteins...termed the dissociation constant or Kd, and its concentration. The smaller the Kd, the more likely molecules...

...poster that Cas9 uses to track down its suspect—the target DNA. tracrRNA: A “trans-activating CRISPR RNA”...target and nontarget DNA strands upstream of the PAM site found in the nontarget strand. Protein Cas: ...sequences and keeping the array organized. Target strand: The strand of foreign DNA that Cas proteins recognize...recognize using an RNA guide. Non-target strand: The paired strand of foreign DNA cut by Cas9, whose sequence...biologists. An sgRNA combines a crRNA and a tracrRNA into a single strand that folds back on itself, simplifying...Regularly Interspaced Short Palindromic Repeats. That contrast of simplicity and complexity is reflected in the... of CRISPR. The bacterial chromosome encodes a tracrRNA (in some systems including Cas9), Cas proteins...