...zinc finger (ZF) nuclease. Zinc fingers recognize triplets and the FokI nuclease operates as a dimer, cutting...) A TAL effector nuclease (TALEN). Similar in principle to the ZF nuclease, the components of the array...zinc fingers that recognize various nucleotide triplets. With some trial and error, ZF arrays are able...their limitations, however. Not every nucleotide triplet has a corresponding zinc finger, and interactions... targets are confined to sequences composed of triplets with corresponding zinc fingers, potential targetable... bp. TAL effectors have some restrictions (for example, the target must start with a T), but they still...Voytas/Bogdanove Golden Gate TALEN kit. In this example, the final array is composed of 18 TAL effector...

... we will use the example of CRISPR/Cas9 in mammalian cells, many of these principles apply to using CRISPR... process of interest. Do you want to: Generate complete and permanent loss of gene expression or function...major concern, Cas9-gRNA ribonucleoprotein (RNP) complexes are advantageous due to the transient Cas9 expression...transgenic embryos Cas9-gRNA ribonucleoprotein (RNP) complexes Purified Cas9 protein and in vitro transcribed...transcribed gRNA are combined to form a Cas9-gRNA complex, which is delivered to cells using cationic lipids Transient... will depend on your specific application. For example: To activate or repress a target gene using dCas9...nucleotides within the selected target sequence. For example, gRNA targeting sequences containing a G nucleotide...

...sfGFP David Spray 54959 mApple-VE-Cadherin-N-10 Tight Junctions VE-Cadherin mApple Michael Davidson 55165... that has a different emission wavelength. For example, if the marker protein is fused to GFP, you may...with their own unique function. The nucleus, for example, houses the cell’s genetic material. The mitochondria...Fusions to other FPs can be found by searching the complete Michael Davidson Fluorescent Protein Collection...Ciliary Base Chibby1 GFP Ken-Ichi Takemaru 89770 pLenti-EGFP-hChibby1 Mother Centrioles / Ciliary Base ...Base Chibby1 EGFP Ken-Ichi Takemaru 118084 pLenti-EB1-EGFP Microtubules EB1 EGFP Ken-Ichi Takemaru 122871...

... lot. At Addgene, AAV particles are titered by droplet digital PCR (ddPCR) using primers and probes targeting...Vector Core is (as of April 2016) also performed by droplet digital PCR (ddPCR). Quality Control Addgene ensures...specific QC experiments were performed on your lot, please contact us . Full sequencing of the Viral Genome...of the viral capsid proteins VP1, VP2 and VP3. Samples of viral preparations are subjected to polyacrylamide...proteins as a fraction of total protein present in the sample is also determined and used to determine purity...shown relative to the total protein present in the sample. Sterility Viral vector preparations are added ...Lentiviral vectors are titered using a lentiviral droplet digital PCR (ddPCR) titration protocol (modified...

...Nature Protocols 2013). For cloning information, please view: Zhang Lab General Cloning Protocol 237.2 ...SpCas9 Plasmids The CRISPR/Cas system can be implemented in mammalian cells by co-expressing the S. pyogenes...Activation Mediator (SAM) is an engineered protein complex for the transcriptional activation of endogenous...gain-of-function screening. For more information, please visit the Addgene SAM library page . Targeted gene... : Expresses the MS2-P65-HSF1 activator helper complex with 2A GFP 61424 : sgRNA cloning backbone with...vector encoding the MS2-P65-HSF1 activator helper complex with a 2A Hygro resistance marker 61427 : lentiviral... In these mice the CRISPR-Cas9 system can be implemented by delivering Cre and sgRNA to a Cre-dependent...

...and protocols showing examples of AAV strategies for intersectional control. Please see our list of suggested...publicly, but please only share materials that you are comfortable and willing to share. Please only share...information. Experimental protocols or schematics. Examples of research materials to share: In vivo data: ...Detailed protocol: AAV Production in HEK293 Cells Please share materials by uploading to your Dropbox link...contributions and your commitment to open science! Please see the meeting preparation section for how to ...

...cilia, centrosomes, the plasma membrane, lipid droplets, the nucleus, and mitochondria. The activity of...ARF GTPase family, information on each gene, and examples of plasmid types available for each gene. Click...gene name for a full list of plasmids available. Please note that some genes may not yet have plasmids ...request. Family Gene Entrez Gene ID Length (aa) Example Expression Systems GTPase Arf1 375 181 Bacterial... and GEF family, information on each gene, and examples of plasmid types available for each gene. Click...request. Family Gene Entrez Gene ID Length (aa) Example Expression Systems GAP Arfgap1 55738 414 Bacterial...

.... With its rapid development (embryogenesis is complete in ~3 days), short generation time (~3 months)...Highlights of Zebrafish Tools in our Collection CRISPR Please refer to Addgene's CRISPR Guide for a general introduction...expressing Cas9 and empty gRNA backbones. A few examples of useful zebrafish CRISPR resources can be found...visualize and track clones of cells over time. Examples of useful tools for studying cells that share ... Alex Schier Lab Other Tools of Interest A few examples of popular tools to use in zebrafish, ranging ...for studying diseases, are listed below: Lipid droplet reporters - Richard White Lab. An imaging platform...dynamics in vivo. Bimolecular fluorescence complementation (BiFC) tools - Marco Morsch Lab. A vertebrate...

...validated gRNA, based on the Zhang lab's PX458 . Multiple gRNA plasmids may be used. The HDR (homology directed...endogenous genes for the isolation of native protein complexes. This vector serves as a backbone to clone the...your experiments can be found below and in the supplemental materials from the Doyon Lab’s publication: ...targeting the PITCh donor plasmid. Note that the principle of the PITCh system is different from the conventional...Degron Tagging Masato Kanemaki's lab has developed a simple and scalable CRISPR/Cas-based method to tag endogenous...nuclear and cytoplasmic proteins, which can then be depleted very rapidly after the addition of auxin to the...and can be scaled up for systematic editing of multiple genes. Plasmids can be found associated with the...

...cloning grade DNA, please let us know using the Cloning Grade DNA Suggestion Form . Please note that this ...µl (± 5ng/µl) Buffer 10 mM Tris-Cl, pH 8.5 Price Please log in to your account and check the individual..., to order more than 2 µg of a single plasmid, multiple orders would need to be placed through our website...website. Every order will need to be completed independently including the check out and will require separate...I find cloning resources or scientific support? Please see our Addgene Protocols for resources you may...