...Restriction enzyme digestion is commonly used in molecular cloning techniques, such as PCR or restriction...digest typically involves ∼500 ng of DNA, while molecular cloning often requires 1 µg of DNA. The total ... from 10-50 µL depending on application and is largely determined by the volume of DNA to be cut. A typical...will need to use a phosphatase to prevent re-circularization of the vector if you are cloning in an insert...sites . Sometimes enzymes cut sequences which are similar, but not identical, to their recognition sites....website about star activity . If you are digesting a large number of plasmids with the same enzyme(s) (for ...

...ATP (extracellular) ecAT3.10 extracellular ATP sensor (cell surface targeted) Imaging extracellular ATP...measurement of a biomolecule while largely preserving spatial and temporal cellular processes. Note that many ...green, red or blue/green) Imaging intraorganellar Ca(2+) at subcellular resolution using CEPIA. Nat Commun...Yasushi Okada ATP (extracellular) GRAB_ATP1.0/ATP1.0mut biosensor for extracellular ATP (Constitutive ...Aug 20;524(7565):309-14. Richard Youle Osmolarity SED1 osmolarity biosensor, for expression in E. coli ...Eckhard Boles pH Imaging intracellular pH in live cells (pHRed) Imaging Intracellular pH in Live Cells with...cytosol Hypoxia triggers subcellular compartmental redox signaling in vascular smooth muscle cells. Circ...

...both Cas12a and Cas3. Similarly, some Acr proteins are specific to a particular species of CRISPR enzymes... Browse Plasmids: Prime Edit Large Scale Edits CRISPR can generate large-scale genetic changes, including...Cas13a-based molecular detection platform. Based on this non-specific cleavage, a Cas13a-based molecular detection...CRISPR Guide Overview CRISPR Basics Small Edits Large Edits Genome-Wide Screening Cas9 Fusion Tools Cas9...techniques are referred to as CRISPR ( C lustered R egularly I nterspaced S hort P alindromic R epeat) technologies...and adaptability, CRISPR rapidly became the most popular genome engineering approach. Figure 1: Overview...knockout or gene edit; or using Cas9 to generate large genomic deletions or inversions with two gRNA sites...

...PRKCG MTH Spinocerebellar ataxia 30 Frederic Mushinski 8418 pLTR PKC gamma PRKCG Spinocerebellar ataxia ...GFP CMV Spinocerebellar ataxia 27 Tobias Meyer 21204 GFP-N2-PKCgamma PRKCG GFP CMV Spinocerebellar ataxia...GFP CMV Spinocerebellar ataxia 25 Tobias Meyer 21215 GFP-C2gamma PRKCG GFP CMV Spinocerebellar ataxia ...CMV Spinocerebellar ataxia 23 Bernard Weinstein 21237 PKC gamma dNPS PRKCG HA CMV Spinocerebellar ataxia... CMV Spinocerebellar ataxia 21 Bernard Weinstein 21239 PKC gamma DN PRKCG HA CMV Spinocerebellar ataxia...CMV Spinocerebellar ataxia 15 Ryohei Yasuda 112270 CMV-PKCg-mEGFP-N1 PRKCG GFP CMV Spinocerebellar ataxia...GFP CMV Spinocerebellar Ataxia Richard Youle 176464 pVPS13D-I-EGFP VPS13D GFP CMV Spinocerebellar Ataxia...

...well-defined cellular sub-populations has generally been limited to single-component cellular definitions...addition to EYFP and ChR2-EYFP, a large number of additional, validated molecular payloads in the INTRSECT configuration..., Flp, and VCre to enable viral expression of molecular payloads in target populations defined by multiple...Plasmids References The development of powerful molecular reagents for biology have propelled our understanding...enabling combinatorial targeting) is a synthetic molecular targeting strategy that allows adeno-associated...multiple recombinase-expression strategies to define cellular sub-populations of interest, including dual-transgenic...

...overexpress this enzyme at a particular point in bacterial growth? Can I produce large amounts of this protein...thereby limiting the use of cellular resources required for the production of large amounts of protein or high...Plasmids You may also like… Plasmids 101 Addgene's Molecular Biology Guides Plasmids 101: E. coli strains for...questions. Does my transcription factor bind to a particular sequence? Do these two proteins interact? What... tell you much about its function and/or alter cellular activity in a way that’s interesting from an applied...Yellow , Orange , Red , Far-Red , Near-Infrared , Large Stokes Shift , Photoactivatable , Photoconvertible...small molecules, light, temperature, or even intracellular metabolite levels. Different plasmids and, often...

...87427 FBL-mEGFP AICSDP-13 mEGFP Fibrillarin Nucleolus (dense fibrillar component) 87428 MYH10-mEGFP AICSDP...Nucleophosmin Nucleolus (granular component) 133963 UBTF-HaloTag AICSDP-80 HaloTag Nucleolar transcription factor... generate fluorescently tagged proteins of key cellular structures in human cell lines. Depositor...CRISPR Protein Tagging Fluorescent Protein Guide Subcellular Localization Plasmids Protocols The Allen Institute... generate fluorescently tagged proteins of key cellular structures in human cell lines of their choice... to create cell lines with endogenously-tagged cellular structures. Addgene ID Plasmid Allen Institute...mEGFP AICSDP-50 mEGFP Nucleophosmin Nucleolus (granular component) 109120 GJA1-mEGFP AICSDP-43 mEGFP Connexin...

...with sequence similarity 3, member C GS3786, ILEI FAM3D family with sequence similarity 3, member D EF7...fms-related tyrosine kinase 1 (vascular endothelial growth factor/vascular permeability factor receptor)...NR1I1 VEGFA vascular endothelial growth factor A MGC70609, MVCD1, VEGF, VEGF-A, VPF VEGFB vascular endothelial...N-RAS, RASH1 ICAM1 intercellular adhesion molecule 1 BB2, CD54, P3.58 ICAM2 intercellular adhesion molecule...The innate immune system is not specific to any particular pathogen. It includes physical barriers such ...immune response is antigen-specific and includes cellular and humoral immunity. Once an adaptive immune ...HSPA10, LAP1, MGC131511, MGC29929, NIP71 ICAM1 intercellular adhesion molecule 1 BB2, CD54, P3.58 IFI30 interferon...

...different reagents that are available for your particular experiment. Select Your Desired Genetic Manipulation...available to carry out your experiment in your particular model organism. There may not be a perfect plasmid...Knockout Permanently disrupt gene function in a particular cell type or organism without a specific preferred... a specific user-defined sequence change in a particular gene, such as generating a point mutation or ...or Interfere (CRISPRi) Reduce expression of a particular gene(s) without permanently modifying the genome...alleles will not contain the desired edit, as a large percentage of DSBs are still repaired by NHEJ. How... and gel electrophoresis (for HDR or NHEJ): For large deletions or insertions, the region of interest ...

... for clarity and does not indicate a specific relationship. p53 Pathway Color is used for clarity and ...transcription factor and tumor suppressor activated by cellular stress. Cancer...transcription factor and tumor suppressor activated by cellular stresses like DNA damage, oxidative stress, and...damaged cells. The p53 name arose from its observed molecular weight (53 kDa) when run on an SDS-PAGE gel. Cancer...on functional p53 through oligomerization. In particular, the loss of p53’s pro-apoptotic effects is especially...only one functional copy of the p53 gene causes a large reduction in tumor suppression activity, leading...

...Polymerase I, Large (Klenow) Fragment 165522 pOpen-K12polLF (exo-) DNA Polymerase I, Large (Klenow) Fragment...plasmids at Addgene for generating enzymes for molecular biology lab work. Depositor...for enzymes that are considered "workhorses" of molecular biology, including DNA polymerases, RNA polymerases... lab costs and overcome supply chain issues, particularly in areas of the world where enzymes are harder...Polymerase 165553 pOpen-BstpolLF Bst DNA Polymerase, Large Fragment 165562 pOpen-Pwopol Pwo DNA Polymerase ...Polymerase 165563 pOpen-BsupolLF Bsu DNA Polymerase I, Large Fragment 165570 pOpen-P45 P45 165572 pOpen-GBDpol ...

... YouTube . Abstract The prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)... and characterization. This strategy relies on cellular delivery of a pair of chimeric single guide RNAs...absence of deletion, the “deletion band” is often too large to efficiently amplify. Typically use primers at...strategy 3 . Prepare a 50 μl reaction mix: 100 ng circular pX330 vector, 1.0 μl annealed oligos (1 μM; see...identified with the desired deletion and move to larger plate or flask for growth. Validation of Biallelic...genomic deletion efficiency mediated by clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 ...Froger A, Hall JE. J. Vis. Exp . 2007. PubMed . Molecular cloning of PCR products. Finney M, Nisson...

...Alzheimer's disease, through large genome-wide association studies (GWAS) or other large scale studies. Researchers...these millions of patients results in billions of dollars spent each year. Unfortunately, the causes of most...with the disease and implicate a wide range of cellular processes. The vast majority of cases (90-95%)...central nervous system, peripheral nervous system, vesicular brain cells and more. New and Noteworthy: AAV....of fluorescent proteins, including Markers of Subcellular Localization, Optogenetics Plasmids, and Chemogenetics...supports the development of a diverse portfolio of biomolecular tools and emphasizes their rapid and broad dissemination...

...and Packaging Plasmids Popular Lentiviral Envelope and Packaging Plasmids Popular γ-Retrovirus Envelope...virus (such as HIV, murine leukemia virus, and vesicular stomatitis virus G) are packaged as replication-defective...infect permissive cells that express ACE2, the cellular receptor for SARS-CoV-2, in a single round of ...particles Murine leukemia-based retroviral particles Vesicular stomatitis virus particles See Addgene's Viral...packaging plasmids. Use the search bar to find a particular viral spike protein (MERS, CoV-1, CoV-2), modification...

...Assays Selection Validation Affinity reagents are molecular tools used in the lab to specifically and tightly...above are used to create hybridomas, which produce large amounts of a single antibody with little variation...antibody is made up of four protein chains: two large heavy chains and two small light chains. These four...In IHC, tissue sections are assayed and the extracellular matrix remains intact. The signaling molecule...enzyme. In ICC, cells have most or all of their extracellular matrix removed. Again, the signaling molecule... fluorophore colors in flow cytometry, but the largest panels used currently are in the mid-forties. When...conjugate or looking for antibodies validated for a similar assay type) and search again. After filtering, ...

...potato) - Scott Lenaghan. Joint Modular Cloning (JMC) Toolkit includes a modular cloning chassis and set of...highlight plasmids spanning a wide variety of molecular biology technologies: from genome engineering ...designed for creating plant expression vectors: A modular plasmid assembly kit for multigene expression, ...multigene constructs - Johannes Stuttmann. MoChlo: Modular Cloning Chloroplast Toolbox for design of synthetic...window) - TAIR includes a database of genetic and molecular biology data for Arabidopsis thaliana research...

...acceptor molecule (also referred to as intermolecular or bimolecular FRET) or (2) conformational changes ...the same protein (also referred to as intramolecular or unimolecular FRET). Fluorescent proteins can be...Protein Resources: Empty Backbones Biosensors Subcellular localization Optogenetics Background Förster ... Constructs to target mTurquoise2 to various subcellular compartments pCEP4CyPet-MAMM Cyan Mammalian Expresses...

...and transparency of the embryo and larvae, it is easy to culture large populations that are amenable to ...reporters, biosensors, and Cre expression plasmids. Large collections or kits are described below . ID Plasmid...Lab Lineage Tracing Cell lineage tracing in multicellular systems typically relies on genetic markers,... Lab Other Tools of Interest A few examples of popular tools to use in zebrafish, ranging from optogenetics...imaging platform to study lipid dynamics in vivo. Bimolecular fluorescence complementation (BiFC) tools - Marco...

...be lysed or the substrate must be delivered intracellularly in order to measure luminescence. Gaussia luciferase...luciferase are less-than-ideal protein tags due to their large size. NanoLuc® Luciferase is small, bright, and ...protein is available – these attributes have made it popular with scientists since its release. Luciferase-containing...Resonance Energy Transfer (BRET) CalFlux VNT : An intracellular calcium sensor created by fusing NanoLuc® and...allow for BRET-based in vivo imaging of multiple cellular processes, biosensors, or optogenetic tools . ... (Rluc8) through an Ala-Glu-Leu linker to the circularly permuted Venus fluorophore. Learn more about ...Gateway cloning for expression in plants Andrew Millar 159707 pG20_LUC2_Hyg Firefly Creation of C-terminal...

...genetically engineered cellular receptors that affect signalling pathways within cells. Subcellular Localization...Monitor small biomolecules or other physiological intracellular processes with genetically encoded fluorescent...Guide Use light to detect, measure, and control molecular signals, cells, or groups of cells with either...Fluorescent Proteins from Kurt Thorn and UCSF Molecular Expressions Galleries from Michael Davidson and...