...cloning and expression of recombinant antibodies. Sci Rep. 2014 Jul 30;4:5885. Andrew Beavil Cloning and mammalian...antigen binding sequence to improve binding and reproducibility. Some scientists choose to work with full length...

...performed using the transfer plasmid, a plasmid encoding Rep and serotype-specific Cap genes, and a plasmid encoding...order to generate high quality preparations. After production, all virus preps are titered and subjected to... containing) AAV particles within representative vector preparations was quantified with electron microscopy...typically prepared using endotoxin-free plasmid purification kits. Resources Browse lentiviral preps available...encoding adenoviral helper sequences. AAV preparations are purified by iodixanol gradient ultracentrifugation...purity and titers adequate for in vivo studies. Preparations are then aliquoted and stored at -80 °C. Titer...on viral genomes isolated from the final AAV preparation. Sequencing results are analyzed to confirm the...

... the Doyon Lab’s publication: Dalvai et al. Cell Rep. 2015 . Alternatively, cDNAs can be cloned directly...Zebrafish Xenopus CRISPR Resources CRISPR Guide Viral Preps Protocols gRNA Design Tools CRISPR Blog Posts The... CRISPR 101 post for more information about HDR/repair template design. How to use CRISPR to tag your ...plasmids may be used. The HDR (homology directed repair) donor plasmid with homology arms and selection...transcription factors with FLAG in human cell lines. To repeat the tagging in your system, use the plasmids as...introduces an N- or C- terminal affinity tag (3xFLAG-2xSTREP) on endogenous genes for the isolation of native...the AAVS1 locus. Doyon Tagging Plasmid: 3xFLAG-2xSTREP Construct for integrating at the AAVS1 "safe harbor...

...Reveals Local and Long-Range Circuit Motifs. Cell Rep. 28(13):3450-3461. PubMed (Link opens in a new window...novel population of long-range inhibitory neurons. Preprint (Link opens in a new window) Yu K, Ahrens S, Zhang...

...Mapping Multiprotein Complexes in Human Cells. Cell Rep . 13(3):621-33. PMID: 26456817 Fu Y, Sander JD, Reyon...other hand, if a repair template with homology arms was supplied, the break could be repaired according to...loci. Activate or Repress : dCas9 fused to transcriptional activators or repressors can modulate transcription...mouse, and rat) Bacteria ( E. coli , Streptococcus , Streptomyces , and others) Drosophila Plants (monocots...Clustered Regularly Interspaced Short Palindromic Repeat) sequences were initially discovered in the E. ...Protospacers are separated by short palindromic repeat sequences. (1) The CRISPR array is transcribed ...tracrRNA) with homology to the short palindromic repeat. The tracrRNA helps recruit RNAse III and Cas9 ...

...sequence. The resulting DSB is then repaired by one of two general repair pathways: the efficient but error-prone...directed repair (HDR) pathway. HDR relies on copying DNA from a matching template to accurately repair or fill... NHEJ is a naturally occurring repair pathway and is the primary repair mechanism for DSBs. While this...base excision repair pathway, which fully removes the uridine from the DNA and replaces it with another... tool to activate, repress, visualize, and isolate genes. Activation or Repression of Target Genes Figure...sufficient to repress transcription by blocking initiation. More robust transcriptional repression (CRISPR ...demethylation DNMT3A - repression through cytosine methylation MQ1 - repression through cytosine methylation...

...Tagging Purification Controlled Expression Reporter Plasmids Reporter Plasmids Tagging and Visualization Purification...Plasmids pBAD (arabinose inducible, glucose repressible) His, Strep, mOCR, MBP, with and without SUMO or TEV...expression at a specific level. Return to Top Reporter Plasmids Reporter plasmids can be used to detect events...they activate expression of the reporter gene. Plasmid ID Promoter Reporter PI Purpose pRU Series Various...Purification Controlled Expression Reporter Plasmids You may also like… Plasmids 101 Addgene's Molecular...Bacteria? E. coli are commonly used to store and replicate plasmids of all types, but beyond that, researchers...Plasmids T7-lacO (lactose/IPTG inducible) Flag, His, Strep, mOCR, MBP, GFP, with and without SUMO or TEV cleavage...

...a maxi-prep culture. Prep size may be scaled based on the required DNA yield. Perform maxi-prep for each...colonies and inoculate into a mini-prep culture. Perform mini-prep for each sample and sequence each colony...Blog Posts Addgene is proud to present a video reprint from the Journal of Visualized Experiments (JoVE...clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system may be...DNA segment by non-homologous end joining (NHEJ) repair. Deletions have potential advantages as compared...oligos at a concentration of 100 μM in ddH 2 O. Prepare a 10 μl reaction mix for each guide and its reverse...using a Golden Gate assembly cloning strategy 3 . Prepare a 50 μl reaction mix: 100 ng circular pX330 vector...

...pcDNA3.1(+)_Strep-Lrrk2-Myc LRRK2 Myc, Strep CMV Parkinson's Maik Hintze 161583 pPuro3.1(+)_Strep-Lrrk2-Myc...Myc, Strep CMV Parkinson's Maik Hintze 161584 pPuro3.1(+)_Strep-Lrrk2(D1994S)-Myc LRRK2 Myc, Strep CMV ...pPuro3.1(+)_Strep-Lrrk2(G2019S)-Myc LRRK2 Myc, Strep CMV Parkinson's Maik Hintze 161586 pPuro3.1(+)_Strep-Lrrk2...A11(2-196)-Strep ANXA11 His, Strep, Tev T7 ALS Lalit Deshmukh 196209 GB1-A11(2-196, G38R)-Strep ANXA11 His... His, Strep, Tev T7 ALS Lalit Deshmukh 196210 GB1-A11(2-196, D40G)-Strep ANXA11 His, Strep, Tev T7 ALS... G175R)-Strep ANXA11 His, Strep, Tev T7 ALS Lalit Deshmukh 196212 GB1-A11(2-196, G189E)-Strep ANXA11 His... His, Strep, Tev T7 ALS Lalit Deshmukh 196213 GB1-A11(2-52)-Strep ANXA11 His, Strep, Tev T7 ALS Lalit ...

...While our repository serves the needs of many researchers, scientists sometimes require preps beyond our...service with all-in-one pricing for viral vector preps, which includes MTA facilitation, DNA amplification...protection levels. We offer 961 in-stock viral vector preps, delivering high quality and ready availability ...start experiments as soon as they receive their AAV prep from Addgene — eliminating the steps to produce,...recombinase, and biosensor transfer plasmids in our repository are eligible for this service. Select any eligible...eligible plasmid in the Addgene repository in your choice of: serotype (AAV1, AAV2, AAV5, AAV8, or AAVrg...ordered in three sizes. For all viral vector preparations, we set both minimum standards and typical titers...

...pTETRIS-cargo Firefly For use as a reporter of the ability of a given lncRNA to cis-repress Mauro Calabrese 106253...Multiple Multi-luciferase reporter vector including transcriptional reporters for NF-kb, TGF-b, c-Myc, ...transcriptional regulation and bioluminescent reporters. Plasmid...Highlights Empty Backbones Expression Constructs Reporter Constructs Luciferase is the enzyme responsible... common choice in assays ranging from use as a reporter gene in vitro and in vivo to use in biosensors...-lives, making them useful as transcriptional reporters. However, they are not secreted, so cells must...CMV-LUC2CP/intron/ARE, CMV-LUC2CP/ARE : A splicing reporter that contains an intron in the middle of the Firefly...

... RNA editing, gene replacement therapy, RNA trans-splicing, and protein replacement to reverse the impacts...suggested roles in transcriptional repression, activity dependent de-repression, chromatin remodeling, gene ...Cell Line Repositories (Link opens in a new window) Coriell Institute - cell line repository, including...in a new window) MMRRC Repository - NIH-supported mouse and cell line repository (Link opens in a new window...largest biorepositories (Link opens in a new window) ATCC - the world's largest cell line repository Tissue...spoken language and hand skills, development of repetitive hand stereotypies, and gait dysfunction or absence...to bind to methylated DNA and recruit known co-repressors or other transcriptional modifiers suggests the...

.... When a repair template is present, the cell may repair a DSB using homology-directed repair (HDR) instead...be used with a repair template to introduce specific edits via homology-directed repair (HDR). ID Plasmid...gRNA-defined target sequence. DSBs are preferentially repaired in the cell by non-homologous end joining (NHEJ...genomic changes, researchers use ssDNA or dsDNA repair templates with homology to the DNA flanking the...Zebrafish Xenopus CRISPR Resources CRISPR Guide Viral Preps Protocols gRNA Design Tools CRISPR Blog Posts Base...base editors convert adenine to inosine, which is replaced by guanosine to create A->G (or T->C on the opposite...nicks create a double-strand break (DSB) that is repaired using error-prone non-homologous end joining (...

.... When a repair template is present, the cell may repair a DSB using homology-directed repair (HDR) instead...be used with a repair template to introduce specific edits via homology-directed repair (HDR). ID Plasmid...Zebrafish Xenopus CRISPR Resources CRISPR Guide Viral Preps Protocols gRNA Design Tools CRISPR Blog Posts The...gRNA-defined target sequence. DSBs are preferentially repaired in the cell by non-homologous end joining (NHEJ...genomic changes, researchers use ssDNA or dsDNA repair templates with homology to the DNA flanking the...base editors convert adenine to inosine, which is replaced by guanosine to create A->G (or T->C on the opposite...nicks create a double-strand break (DSB) that is repaired using error-prone non-homologous end joining (...

...generate TALORs (TAL Orthongal Repressors) that can be used to custom repress gene expression in yeast. TALORs... customizable arrays of polymorphic amino acid repeats in the TAL effectors. View Addgene's TALEN Guide...efficiently assemble TALEN constructs with custom repeat arrays. TALEN Kits TALEN Kit Description Depositing...along with strong nuclear localisation tags and repress transcription initiation when targeted to DNA sequences... pFUS array vectors, destination vectors and a reporter vector for mammalian cell-based validation assay...N- and C-terminal domains (N153AA, C47AA). The reporter vector pGL4-SSA can be used for single-strand ... TALEs), 63 wild-type TAL amino acids after the repeat domain, and C-terminal SV40 NLS and HA tags. Plasmid...

..., Addgene is distributing ready-to-use viral preparations in engineered systemic serotypes from the Viviana...and full sequencing of the final viral vector preparation. Systemic Delivery AAV Capsid Choice Need help...Addgene’s Data Hub and the Caltech CLOVER Center’s repository of published capsid use (Link opens in a new ...: Systemic Capsids PHP.eB These viral vector preparations were produced with the pUCmini-iCAP-PHP.eB plasmid...tropism exhibited by the PHP.eB serotype has been reported to occur by using the cellular receptor LY6A (...expression iCre AIBS , Ting PHP.S These viral vector preparations were produced with the pUCmini-iCAP-PHP.S plasmid...tdTomato Control Boyden PHP.V1 These viral vector preparations were produced with the pUCmini-iCAP-PHP.V1 plasmid...

...tetracycline-dependent repression. Mutating these residues led to the development of a reverse Tet repressor, or rTetR...derived from pCW57.1. rtTA was replaced with tTA, and Puromycin was replaced with Blasticidin selection. ...to be replaced by the gene of interest. tTA Off Sabatini 92099 AAVS1_Puro_Tet3G_3xFLAG_Twin_Strep Bidirectional...regulated inducible transcriptional activation or repression Plasmid... the Tet Response Element (TRE) A TRE is seven repeats of a 19 nucleotide tetracycline operator (tetO)...sequence, and is recognized by the tetracycline repressor (tetR). In the endogenous bacterial system, if...presence of tetracycline for induction, rather than repression. The new transactivator rtTA ( r everse t etracycline-controlled...

.... When a repair template is present, the cell may repair a DSB using homology-directed repair (HDR) instead...be used with a repair template to introduce specific edits via homology-directed repair (HDR). ID Plasmid...gRNA-defined target sequence. DSBs are preferentially repaired in the cell by non-homologous end joining (NHEJ...genomic changes, researchers use ssDNA or dsDNA repair templates with homology to the DNA flanking the...Zebrafish Xenopus CRISPR Resources CRISPR Guide Viral Preps Protocols gRNA Design Tools CRISPR Blog Posts Base...base editors convert adenine to inosine, which is replaced by guanosine to create A->G (or T->C on the opposite...nicks create a double-strand break (DSB) that is repaired using error-prone non-homologous end joining (...

.... When a repair template is present, the cell may repair a DSB using homology-directed repair (HDR) instead...be used with a repair template to introduce specific edits via homology-directed repair (HDR). ID Plasmid...Zebrafish Xenopus CRISPR Resources CRISPR Guide Viral Preps Protocols gRNA Design Tools CRISPR Blog Posts The...gRNA-defined target sequence. DSBs are preferentially repaired in the cell by non-homologous end joining (NHEJ...genomic changes, researchers use ssDNA or dsDNA repair templates with homology to the DNA flanking the...base editors convert adenine to inosine, which is replaced by guanosine to create A->G (or T->C on the opposite...nicks create a double-strand break (DSB) that is repaired using error-prone non-homologous end joining (...

...example: To activate or repress a target gene using dCas9-activators or dCas9-repressors, gRNAs should be targeted...region where the edit should be made HDR requires a repair template and displays reduced efficiency compared...Base editors can make a limited set of mutations. Repress or Interfere (CRISPRi) Reduce expression of a particular...without permanently modifying the genome dCas9-repressor (such as dCas9-KRAB) or dCas9 gRNA(s) targeting...promoter is typically used for gRNA May contain reporter gene (e.g. GFP) to identify and enrich positive...vector or separate transfer vectors May contain reporter gene (e.g. GFP) or selection marker to identify...CRISPR protocols from Addgene depositors JOVE Video Reprint: Protocol for genomic deletions in mammalian cell...