...expressing Rep2 and PHP.eB Cap controlled by tTA-TRE amplification system Gradinaru 103006 pUCmini-iCAP-PHP.S PHP.S...expressing Rep2 and PHP.S Cap controlled by tTA-TRE amplification system Gradinaru 127847 pUCmini-iCAP-PHP.V1 ...expressing Rep2 and PHP.V1 Cap controlled by tTA-TRE amplification system Gradinaru 175004 pUCmini-iCAP-AAV.CAP-B10...expressing Rep2 and B10 Cap controlled by tTA-TRE amplification system Gradinaru 175005 pUCmini-iCAP-AAV.CAP-B22...expressing Rep2 and B22 Cap controlled by tTA-TRE amplification system Gradinaru 185136 pUCmini-iCAP-AAV.MaCPNS1... Rep2 and MaCPNS1 Cap controlled by tTA-TRE amplification system Gradinaru 185137 pUCmini-iCAP-AAV.MaCPNS2... Rep2 and MaCPNS2 Cap controlled by tTA-TRE amplification system Gradinaru RepCap Plasmids for Serotypes...

...nuclease form a complex with each individual, unique crRNA. (4) Each crRNA:tracrRNA:Cas9 complex seeks out ...Anti-CRISPR genes employ varied mechanisms to block CRISPR. Some prevent the CRISPR-Cas complex from binding...a protospacer adjacent motif (PAM). The CRISPR complex binds to the foreign DNA and cleaves it to destroy...out the DNA sequence complementary to the crRNA. In Type II CRISPR systems, a potential target sequence ...after where the crRNA would bind. (5) After the complex binds, Cas9 separates the double stranded DNA target...strands near the PAM. (6) The crRNA:tracrRNA:Cas9 complex unbinds after the double-strand break. CRISPR systems...classes based on the structure of the effector complex that cleaves the genomic DNA. Type II CRISPR was...

...receptor GPR38, MTLR1 MPL myeloproliferative leukemia virus oncogene C-MPL, CD110, MPLV, TPOR MSTN myostatin...histocompatibility complex, class I, A FLJ26655, HLAA HLA-B major histocompatibility complex, class I, B AS...histocompatibility complex, class II, DM beta D6S221E, RING7 HLA-DOA major histocompatibility complex, class II...histocompatibility complex, class II, DO beta DOB HLA-DPA1 major histocompatibility complex, class II, DP ...histocompatibility complex, class II, DP beta 1 DPB1, HLA-DP1B HLA-DQA1 major histocompatibility complex, class II...histocompatibility complex, class II, DQ alpha 2 HLA-DXA HLA-DQB1 major histocompatibility complex, class II, ...histocompatibility complex, class II, DR alpha HLA-DRA1 HLA-DRB1 major histocompatibility complex, class II, ...

...CRISPR in your research. Although we will use the example of CRISPR/Cas9 in mammalian cells, many of these... process of interest. Do you want to: Generate complete and permanent loss of gene expression or function... the edit should be made HDR requires a repair template and displays reduced efficiency compared to NHEJ...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...

...SpCas9 Plasmids The CRISPR/Cas system can be implemented in mammalian cells by co-expressing the S. pyogenes...codon-optimized SpCas9; sgRNA can be co-transfected as PCR amplicons (Ran et al., Nature Protocols 2013). Individual...Activation Mediator (SAM) is an engineered protein complex for the transcriptional activation of endogenous... : 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...SaCas9; SaCas9 sgRNA can be co-transfected as PCR amplicons using a protocol similar to SpCas9 sgRNA PCR (... In these mice the CRISPR-Cas9 system can be implemented by delivering Cre and sgRNA to a Cre-dependent...

...for the 20 µL sized testing sample is denoted with a .T suffix (for example, AAV1.T). The catalog ID for...for the 100 µL sized sample does not have the suffix (for example, AAV1). AAV Vectors for Serotype Testing...different serotypes. In addition to the standard 100 µL sample size, the vectors listed below are also available...production page . Note: For a list of all 100 µL sized sample, please see our Control AAV Inventory . The catalog...include virus associated DNA (vdna), which is a sample of the purified DNA from the original plasmid that...

... kinase, is found in two complexes, mTORC1 and mTORC2. Each complex contains a unique subunit...links to each gene page are provided below. mTORC1 Complex Symbol Name 4E-BP Eukaryotic translation initiation... RPTOR; Regulatory associated protein of MTOR, complex 1 Rheb Ras homolog enriched in brain S6K Also known...Hippel-Lindau tumor suppressor Return to top mTORC2 Complex Symbol Name AKT AKT1 AKT2 AKT3 v-akt murine thymoma...homolog Rictor RPTOR independent companion of MTOR, complex 2 SGK Serum/glucocorticoid regulated kinase 1 Return...

... genomic DNA. This DNA is then used as a template to amplify a portion of the intended lentiviral insert...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 ...rigorous barcode matching system that follows the sample from the DNA tube all the way to the cleared viral...

...in the following articles provide PCR templates for amplification of the tag (eg GFP, Flag, YFP, etc) and...101 post for more information about HDR/repair template design. How to use CRISPR to tag your gene of ...endogenous genes for the isolation of native protein complexes. This vector serves as a backbone to clone the...encodes both a guide RNA transcript and a repair template for an individual tagging event. The kit contains...Degron Tagging Masato Kanemaki's lab has developed a simple and scalable CRISPR/Cas-based method to tag endogenous...

... you'll need Sanger sequencing reads from PCR amplicons that cover your locus of interest and correspond... from a deep sequencing experiment a reference amplicon sequence to assess and quantify the efficiency...efficiency of the targeted mutagenesis The amplicon sequence expected after HDR can be provided as an optional...CRISPResso suite accommodates single or pooled amplicon deep sequencing, WGS datasets and allows the direct...for NmCas9 and BsmBI site for cloning in gRNA: pSimpleII-U6-tracr-U6-BsmBI-NLS-NmCas9-HA-NLS(s) PDF 47.5...-EGFP PDF 2.3 MB Zhang GeCKO pooled library amplification 1 vector system: lentiCRISPR v2 2 vector system...

...total protein content of the sample is determined. Only antibody samples with >90% purity are distributed...using a NanoDrop spectrophotometer. If needed, the sample is adjusted by diluting or concentrating to a final...Coomassie staining. For Coomassie staining, the sample is separated via a denaturing SDS-PAGE gel alongside...transfection. This residual plasmid is extracted and amplified with primers targeting the heavy chain and/or ...

...including legal approval and an implementation letter DNA amplification Viral vector production Density...preps, which includes MTA facilitation, DNA amplification, and high-quality quality control measures such... Quality is our priority. During this time, we amplify DNA, produce the prep, and perform comprehensive...researchers with troubleshooting and results that complement published data, capturing the insights that often...

.... With its rapid development (embryogenesis is complete in ~3 days), short generation time (~3 months)...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 ...dynamics in vivo. Bimolecular fluorescence complementation (BiFC) tools - Marco Morsch Lab. A vertebrate...

...changes, researchers use ssDNA or dsDNA repair templates with homology to the DNA flanking the DSB and ...edit close to the gRNA PAM site. When a repair template is present, the cell may repair a DSB using homology-directed...Nickase mutants can also be used with a repair template to introduce specific edits via homology-directed...primer binding site and the desired edits on an RT template. ID Plasmid Gene/Insert Promoter Selectable Marker...or cloning method. To isolate mammalian vectors, simply type "Mammalian" into the search bar. When using...

...changes, researchers use ssDNA or dsDNA repair templates with homology to the DNA flanking the DSB and ...edit close to the gRNA PAM site. When a repair template is present, the cell may repair a DSB using homology-directed...Nickase mutants can also be used with a repair template to introduce specific edits via homology-directed...primer binding site and the desired edits on an RT template. ID Plasmid Gene/Insert Promoter Selectable Marker...none S. pyogenes Kamoun 52255 pUC119-gRNA U6 PCR template none S. pyogenes Sheen 51295 pRGEB31 rice snoRNA...

... 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...

...Collection INTRSECT Plasmid Collection INTRSECT Implementation Plasmids References The development of powerful...populations defined by multiple parameters. Figure 1: Examples of intersectional cell population definitions ...order to enable three-feature cell targeting. C) Example of viral targeting of cells expressing Cre AND ...producing a functional molecular tool (C,F). Implementation The following resources may be of interest ...interest for groups interesting in implementing intersectional experimental design. The Deisseroth Lab maintains...

...parts can be combined to construct complex networks. This is accomplished through the use of assembly standards...meaning that the suffix will need to be reverse complimented for primer design. The restriction enzymes used...encodes Gly-Ser in-frame with prefix start codon; Employs high-efficiency enzymes. Prohibited Restriction...

...pAAV-synP-FLEX-splitTVA-EGFP-B19G Can be used to complement deletion-mutant rabies virus AAV1 Wickersham ...pAAV-syn-FLEX-splitTVA-EGFP-tTA These AAV together can be used to complement deletion-mutant rabies virus AAV1 Wickersham ...lacks the ability to infect neighboring cells. For example, G-deleted rabies lacks the viral glycoprotein...infect any neighboring cells. Thus, through complementation, G-deleted rabies virus can spread to monosynaptically...

...changes, researchers use ssDNA or dsDNA repair templates with homology to the DNA flanking the DSB and ...edit close to the gRNA PAM site. When a repair template is present, the cell may repair a DSB using homology-directed...Nickase mutants can also be used with a repair template to introduce specific edits via homology-directed...primer binding site and the desired edits on an RT template. ID Plasmid Gene/Insert Promoter PI Publication...