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...of Pennsylvania Vector Core. Transfections are performed using the transfer plasmid, a plasmid encoding...are purified by iodixanol gradient ultracentrifugation and concentrated to purity and titers adequate for...cell cultures are inspected for sterility. Transducibility Some viral vectors are tested in vitro and ...section for images). Figure 2: AAV Pro cells were transduced with either pAAV-Ef1a-mCherry-IRES-Cre (55634...cleared by low-speed centrifugation and then by filtration through a 0.45 µm polyethersulfone (PES) membrane... been stored at -80 °C and thawed. Lentivirus titration is performed using a 293T cell line in the presence...using a lentiviral droplet digital PCR (ddPCR) titration protocol (modified from Wang et al. (2018) (Link...

...information on substrates and synthases needed for use of these tags. Plasmids Addgene ID Plasmid Substrate Feature...simultaneously with dual labeling when used with two substrates Can be used in live or fixed cells, and for internal... proteins and use enzymatic binding to attach substrate Control plasmids with a recognizable sub-cellular... is based on the human O6-alkylguanine-DNA-alkyltransferase (hAGT), a DNA repair protein, and is suitable...protein tags based on the acyl carrier protein. Substrates are derivatives of Coenzyme A (CoA). These tags...Vector SNAP-tag Empty backbone for stable and transient mammalian expression 101133 pSNAP-CaaX Control...Vector CLIP-tag Empty backbone for stable and transient mammalian expression 101134 pCLIPf-H2B Control...

... of the CRISPR RNA (crRNA) and the trans-activating crRNA (tracrRNA; Hsu et al ., Nature Biotechnology...plasmids #61422 - #61424 were designed for transient transfection : 61422 : Expresses dCAS9-VP64 activator... cassettes to express the CRISPR RNA array and tracrRNA separately (Cong et al ., Science 2013). Additionally...or SpCas9n, D10A nickase) + CRISPR RNA array + tracrRNA: This plasmid contains three expression cassettes...human codon-optimized SpCas9; sgRNA can be co-transfected as PCR amplicons (Ran et al., Nature Protocols...references are below. Return to top Cas9 SAM transcriptional activation plasmids and screening library CRISPR...) is an engineered protein complex for the transcriptional activation of endogenous genes. It consists...

...Resonance Energy Transfer (FRET) is a process by which energy is non-radiatively transferred from an excited...Quantitative multiphoton spectral imaging and its use for measuring resonance energy transfer mCerulean, mVenus... energy transfer to multiple acceptors Koushik et al. (2009) Anomalous surplus energy transfer observed...fluorescent proteins, and other molecules have been demonstrated for use in FRET experiments. FRET efficiency...FLIM) or FLIM-FRET, where the increased energy transfer through FRET reduces the donor lifetime. FRET ...fused to the same protein (also referred to as intramolecular or unimolecular FRET). Learn more about FRET...integral of donor emission and acceptor excitation spectra (×10 15 M -1 cm -1 nm 4 ). Values obtained from...

...easily and inexpensively generate R-mAbs by transiently transfecting the plasmids into mammalian tissue culture... Anti-Pan-Neurofascin (extracellular) [A12/18R] Pan-Neurofascin (extracellular) Rat Mouse IgG2a 177439...Anti-Kv1.4 K+ channel (extracellular) [L71/5R] Kv1.4 K+ channel (extracellular) Rat Mouse IgG2a 177489...Mouse IgG2a 177564 Anti-ADAM22 (extracellular) [N57/2R] ADAM22 (extracellular) Mouse Mouse IgG2a 177565 Anti-GluN2B...channel Rat Mouse IgG2a 188161 Anti-TRAAK K+ channel [N445/27R] TRAAK K+ channel Mouse Mouse IgG2a 188162...Anti-Kv1.4 K+ channel (extracellular) [L71/22] Kv1.4 K+ channel (extracellular) Rat Mouse IgG2a 190292...N382/14] MFF Human Mouse IgG2a 190320 Anti-TRAK2 [N390/43] TRAK2 Human Mouse IgG2a 190321 Anti-Navbeta2 Na...

... by Iodixanol Gradient Ultracentrifugation AAV Titration by ddPCR AAV Titration by qPCR Using SYBR Green...use three plasmids: A transfer plasmid (or cis plasmid) containing the transgene or genetic elements of...service gives you even more options. AAV Transfer Plasmids Transfer plasmids encode the genetic information...Browse AAV transfer, packaging, and helper plasmids in Addgene's repository. Viral...Plasmids Adeno-Associated Viral (AAV) Plasmids AAV Transfer Plasmids AAV Packaging Plasmids AAV Helper Plasmids...This collection provides a curated selection of transfer, packaging, and helper plasmids that can be used... This table contains a general list of all the transfer plasmids in Addgene's repository. You can search...

...samples to extract proteins. Homogenize (tissues only), lyse, and then collect protein extract by centrifugation...hybridoma cells into plasmids. Plasmids are then transfected into cells and induced to express antibodies....to lot, as immune responses have high inter- and intra-individual variability. Other Affinity Reagents ...and therefore should be kept at 4 °C. Record and track lot numbers for all antibodies used in the lab. ...reaction in the presence of several different substrates. The luminescence produced by the reaction can... an SDS-PAGE gel to separate proteins by size. Transfer proteins from the gel to a membrane. Incubate ...and block the membrane. Activate the HRP with a substrate kit. Image the membrane using either X-ray film...

...MECP2 disorders. The RSRT has a comprehensive, strategic and aggressive plan to use gene editing, MECP2...reactivation, RNA editing, gene replacement therapy, RNA trans-splicing, and protein replacement to reverse the...purposeful hand movements and regression of speech. In contrast to the sustained loss of skills in neurodegenerative...disease severity. The MECP2 protein is a global transcriptional regulator of thousands of genes and studies...studies have suggested roles in transcriptional repression, activity dependent de-repression, chromatin remodeling...DNA and recruit known co-repressors or other transcriptional modifiers suggests the major function of MECP2...missense mutations cluster in the MBD and NID demonstrating the importance of binding to methylated DNA ...

... E2F E2F1 E2F2 E2F3 E2F transcription factor ECT2 Epithelial cell transforming 2 EGFR Epidermal growth...family. GTPases serve as molecular switches that transmit signals within the cell. GTPases have an active...EIF4EBP EIF4EBP1 EIF4EBP2 EIF4EBP3 Eukaryotic translation initiation factor 4E binding protein ERBB2 Erb-b2...protein kinase ETS ETS1 ETS2 ETS proto-oncogene, transcription factor EXOC EXOC1 EXOC2 EXOC3 EXOC4 EXOC5 EXOC6... related tyrosine kinase 3 FNT FNTA FNTB Farnesyltransferase, CAAX box FOS FBJ murine osteosarcoma viral...protein 2 ICMT Isoprenylcysteine carboxyl methyltransferase INSR Insulin receptor INSRR Insulin receptor-related...receptor-related receptor IRS IRS1 IRS2 Insulin receptor substrate JUN Jun proto-oncogene KSR KSR1 KSR2 Kinase suppressor...

... Flp, here expressed through a double transgenic animal strategy (PV-2a-Cre; SOM-IRES-Flp). Image from...recombinase-expression strategies to define cellular sub-populations of interest, including dual-transgenic recombinase-expressing...Ansorge M, Awatramani R, Kang UJ, Rayport S. 2018. Dopamine neuron glutamate cotransmission evokes a delayed... AND Flp, Cre NOT Flp, or Flp NOT Cre. B) This strategy has been expanded to incorporate VCre in order... targeting) is a synthetic molecular targeting strategy that allows adeno-associated virus (AAV)-borne...recombinase-expressing mouse lines 2–9 and combinations of transgenic recombinase-expressing animal lines and retro-grade...in a new window) of Flp-recombinase-expressing transgenic mouse and rat lines. The Fenno Lab maintains ...

...in vitro transcription as an alternative. Based on transient double-selection of transfected cells, the...new tools for genome engineering. Fusions of transcription activator-like (TAL) effectors of plant pathogenic...comprised of 834 plasmids of pre-assembled trimer and tetramer RVD domains designed for rapid TALEN assembly....and Chad Cowan Ekker Lab TALEN Kit All possible tetramers (264) of RVDs, compatible with Golden Gate TALEN...strong nuclear localisation tags and repress transcription initiation when targeted to DNA sequences within...Gate-assembled TALEN, both in microinjected embryos and transfected cells. TALEN expression is driven by the strong...reporter vector pGL4-SSA can be used for single-strand annealing assay, which enables the evaluation of...

...editing to easily engineer specific transgenic worm lines. The transparent body of the C. elegans hermaphrodite...Caenorhabditis elegans. Mos1 Based Transgenesis miniMos Transgenesis Kit - Erik Jorgensen Lab. This kit...consists of 959 somatic cells, which makes them attractive for imaging and fluorescence microscopy experiments...along with links to protocols, can be found below: SapTrap-SEC kit for C. elegans genome engineering - Bob...kit can be found at the Goldstein Lab website . SapTrap CRISPR/Cas Toolkit - Erik Jorgensen Lab. A collection...using CRISPR/Cas9 and the selection of C. elegans strains. CRISPR plasmids from John Calarco's lab as described...Craig Mello's lab and described in A Co-CRISPR Strategy for Efficient Genome Editing in Caenorhabditis...

...NanoLuc fusions are useful for both intracellular and extracellular applications where you want minimal...HaloTag, SmBiT, and LgBiT, useful for detecting and tracking key drug target proteins. Depositor... educational resources, and efficient material transfer solutions that help advance science while reducing...analysis and protein fusion vectors for detecting and tracking proteins using tags such as NanoLuc, HaloTag, ...Promega collection are available in two formats: pretransformed in bacteria as an agar stab 20 µg purified ...signaling pathways, investigating RNA regulation, tracking protein degradation, analyzing CRISPR-mediated...bright luminescent signal in the presence of a substrate. NanoBiT PPI assays enable kinetic, real-time ...

...Vector Core Viral Vectors Lentiviral Transfer Vectors Adenoviral Transfer Vectors Resources Kazuhiro Oka and...lentiviral and adenoviral transfer vectors with Addgene. Lentiviral Transfer Vectors Within this set of...packaging these transfer vectors and generating new viral vectors. Download the Lentivirus Transfer Vector Production...from the PGK promoter Adenoviral Transfer Vectors The adenoviral transfer vectors contained within this ... of lentiviral transfer vectors you can find reporter plasmids to test whether or not you've efficiently...coexpressed with tdTomato and Pac (puromycin N-acethyl-transferase) pCDH-CB-copGFP-T2A-iCre 72256 Expresses copGFP...Flp recombinases. Genes can be cloned into these transfer vectors using shuttle vectors pICPIS-EF1 (Addgene...

...Browse the Zhang lab’s comprehensive human transcription factor collection, available at Addgene. The...The collection can be used for screening transcription factors for cellular programming or cancer drug ...Library The MORF library is a comprehensive human transcription factor collection that contains 1,836 genes ...collection, can be purchased at Addgene. Each transcription factor isoform has a unique 24 bp barcode that...that simplifies identification of the transcription factor isoform in pooled screens. This collection ...collection and library can be used for screening transcription factors for applications such as cellular programming...cancer drug resistance. Protocols & References A transcription factor atlas of directed differentiation. Joung...

... to strain-specific tropism (See S3 table (Huang, et al., 2019) for LY6A-permissive mouse strains). AAV-PHP.eC... not display strain-specific tropism and can be used in LY6A-nonpermissive mouse strains. **AAV1-X1 has....AX with GRE or transgenic animal Oligodendrocytes - AAV-PHP.eB with GRE or transgenic animal Brain vascular....AX with GRE or transgenic animal L5 & Inhibitory - AAV-PHP.eB with GRE or transgenic animal L2/3 & L4...AAV.CAP-B10 with GRE or transgenic animal Excitatory - AAV.PHP.N with GRE or transgenic animal For PNS applications...several factors including: the delivery method, mouse strain, target cell type or tissue of interest, and model...

...pcDNA3-HtrA2-EGFP HTRA2 GFP CMV Parkinson's L. Miguel Martins 14122 pcDNA3-HtrA2-EGFP S306A HTRA2 GFP CMV...pcDNA3-delta133HtrA-EGFP HTRA2 GFP CMV Parkinson's L. Miguel Martins 14126 pET20b-delta133HtrA2 HTRA2 His...pcDNA3-HtrA2-FLAG HTRA2 Flag CMV Parkinson's L. Miguel Martins 15939 pcDNA3-HtrA2-FLAG S142D HTRA2 Flag ...puro HTRA2 sg1 HTRA2 hU6 Parkinson's Alexis Jourdain 244864 pLentiCRISPR V2 puro HTRA2 sg2 HTRA2 hU6 Parkinson's...pcDNA3-HtrA2-FLAG S400D HTRA2 Flag CMV Parkinson's L. Miguel Martins 16153 pET20b-delta133HtrA2 S142A ...S142A HTRA2 His T7 Parkinson's L. Miguel Martins 16154 pET20b-delta133HtrA2 S142D HTRA2 His T7 Parkinson's...pET20b-delta133HtrA2 S400A HTRA2 His T7 Parkinson's L. Miguel Martins 16156 pET20b-delta133HtrA2 S400D ...

... Dorus Gadella 58473 pEGFP-C1 F-tractin-EGFP Actin filaments F-tractin EGFP Dyche Mullins 41147 pEGFP ...Atsushi Miyawaki 212019 pcDNA3/F-tractin=mStayGold Actin filaments F-tractin mStayGold Atsushi Miyawaki 229608...Membrane Trafficking The secretory pathway is responsible for delivering proteins to the extracellular environment... Proteins are first translated at the endoplasmic reticulum, then are transported to the Golgi apparatus...Localization General Cytoskeleton Junctions Membrane Trafficking Additional Resources The subcellular localization...observe cytoskeleton remodeling or blocked membrane traffic. Addgene has assembled a collection of popular ...find markers for components of these membrane trafficking pathways and structures. ID Plasmid Structure...

...of the applicable transfer plasmid. We will help you gain legal usage of the transfer plasmid from the ...and useful gene delivery tools that effectively transduce many mammalian cell types. AAV is used in a wide...service lets scientists choose from over 4,500 transfer plasmids to be packaged in their choice of eight...chemogenetic, optogenetic, recombinase, and biosensor transfer plasmids in our repository are eligible for this...aliquots 1.0 mL Large 25 × 100 µL aliquots 2.5 mL Extra Large 50 × 100 µL aliquots 5.0 mL *AAV2 yields are...several important steps: MTA facilitation for the transfer plasmid, including legal approval and an implementation...experiments. Shipping 2–7 days We will email you with tracking information as soon as your order ships. Shipping...

...into human FBL locus Jorgensen Lab SapTrap CRISPR/Cas Toolkit SapTrap is a modular toolkit from Erik Jorgensen's...Genome Editing Cut Base Edit Nick Prime Edit Transcriptional Regulation Activate Interfere Epigenetics RNA... for improved efficiency and throughput over traditional tagging methods. Several different tagging techniques...CRISPR-Cas tagging system were tested by tagging transcription factors with FLAG in human cell lines. To repeat...insert genetic tags in the C. elegans genome. The SapTrap reaction produces a single plasmid targeting vector...vector that encodes both a guide RNA transcript and a repair template for an individual tagging event....26 plasmids; 21 of the plasmids are for use in SapTrap reactions for targeting vector assembly, and the...