...fluorophores to distinguish between cells. Most flow experiments will use one marker to distinguish between live... Minibodies are engineered antibody fragments consisting of two scFvs bound to a CH3 domain. Fab fragments...measurable light or color in response to a specific stimulus, can be conjugated to antibodies in order to allow...machine or by X-ray film. HRP cannot be used to distinguish between different antibodies in the same reaction...the number of signaling molecules that can be distinguished in your application. The indirect detection ...molecule to epitope ratio. Secondary antibodies can distinguish between different species of origin, isotypes...between DNA and protein. Micrococcal nuclease digestion is used to shear the DNA in these assays. This...

...Marrack Labs. Point-of-care testing for COVID-19 using SHERLOCK diagnostics. Joung J, et al. medRxiv 2020.05.04.20091231...credit: Maya Kostman for the Innovative Genomics Institute Addgene COVID-19 Resources The following types...SHERLOCK and DETECTR . Feng Zhang's lab at the Broad Institute has utilized the natural RNAse activity of the...UnLOCKING (SHERLOCK and SHERLOCKv2). The Broad Institute has published information and protocols for utilizing...2019 novel coronavirus SARS-CoV-2 using CRISPR diagnostics: SARS-CoV-2 DETECTR (Link opens in a new window...-CoV-2 detection assay for laboratory and home testing. Kellner, et al. bioRxiv 2020.06.23.166397 (Link...now available from Stanley Qi's lab. COVID-19 Diagnostic Toolkit Enzymes. (unpublished) Taq polymerase...

...experiments Yeast GAL4, PGK, ADH1, ADE2, TRP1 Gateway destination vectors - Inducible expression...Neo DEST - Lentiviral Gateway destination vector for gene expression Puromycin...Puro DEST - Lentiviral Gateway destination vector for gene expression Hygromycin...Hygro DEST - Lentiviral Gateway destination vector for gene expression pLenti...TO Hygro - Lentiviral Gateway destination vector for gene expression (tet...GFP-Zeo DEST - Lentiviral Gateway destination for shRNA expression pLenti CMV... Tet-inducible lentiviral Gateway destination vector for gene expression GFP...

... Standards SynBio Blog Genome Engineering A distinguishing feature of synthetic biology is the ease with...ACTAGT A GCGGCCG CTGCAG Suffix Enzymes: SpeI , NotI , PstI Scar: TACTAGAG or TACTAG Features: 8 bp Scar; No...Prohibited Restriction Sites: EcoRI, XbaI, SpeI, PstI Restriction Sites to Avoid: NotI For more info, ...GCTAGC GCGGCCG CTGCAG Suffix Enzymes: Nhel , NotI , PstI Scar: GCTAGT Features: 6 bp scar encodes Ala-Ser...Prohibited Restriction Sites: EcoRI, SpeI, NheI, PstI Restriction Sites to Avoid: PvuII, XhoI, AvrII, ...ACTAGT A GCGGCCG CTGCAG Suffix Enzymes: SpeI , NotI , PstI Scar: ACTAGA Features: Modified RFC 10 with shortened...Prohibited Restriction Sites: EcoRI, XbaI, SpeI, PstI For more info, visit iGEM: Silver Standard (RFC ...

... the Ras pathway. Ras is a small GTPase and constitutively active Ras is the most common oncogene in human... occur in the Ras gene it can be stuck in a constitutively active form, constantly activating pathways...cell growth and potentially leading to cancer. Constitutively active Ras is the most common oncogene in human...v-akt murine thymoma viral oncogene homolog 3 ALK Anaplastic lymphoma receptor tyrosine kinase ARHGAP35 Rho...MLST8 MTOR associated protein, LST8 homolog MTOR Mechanistic target of rapamycin NF1 Neurofibromin 1 NFE2L2... 1 RALGDS Ral guanine nucleotide dissociation stimulator RAPGEF RAPGEF1 RAPGEF2 Rap guanine nucleotide.... The RAS Initiative - at the National Cancer Institute (NCI) References Ras oncogenes and their downstream...

...pLenti CMV Puro DEST 3rd Gateway destination plasmid with constitutive or inducible expression of cDNAs...lentiviral systems Viral production Frequently asked questions about lentivirus use Glossary of commonly used...plasmid. Nolan 12257 pWPXL 2nd EF-1alpha driven constitutive transgene expression, contains intron that gives...expression. Trono 12254 pWPI 2nd EF-1alpha driven constitutive transgene expression and EGFP coexpression. ...inducible shRNA or cDNA expression, gateway plasmid, constitutive Hygromycin resistance gene coexpression. See...Fraser 15948 pLOVE 3rd Tet inducible gateway destination plasmid for cDNA expression Ramalho-Santos 24129...this plasmid. Root Return to Top Do you have suggestions for other plasmids that should be added to this...

...PMID: 25398340 Walton, R. T., Christie, K. A., Whittaker, M. N., & Kleinstiver, B. P. (2020). Unconstrained...Publications CRISPR Overview Bacteria have an interesting adaptive immune response that detects and eliminates...DNA with no homology elsewhere in the genome. Realistically, most gRNA targeting sequences will have additional..., a large portion of the Cas9-induced DSBs will still be repaired via NHEJ. The resulting population of...integrase landing site ( attB ) and the other to stimulate DNA repair. This can result in more efficient ...activity. Knockout libraries often target 5′ constitutively expressed exons, while activation and repression...repression, many CRISPR epigenetic tools require constitutive expression of the dCas9 fusion and/or expression...

...syndrome is currently based on specific clinical diagnostic criteria, defined as the presence of relatively...gait. Greater than 95% of individuals who meet diagnostic criteria have disease-causing mutations in the...switched on regardless of age or symptom severity, suggesting that restoring MECP2 expression in humans could...that can be differentiated into neurons Coriell Institute: Patient-specific lines (Link opens in a new window... database of clinical features and genetic characteristics of Rett syndrome (Link opens in a new window...Repositories (Link opens in a new window) Coriell Institute - cell line repository, including patient-derived...18337588 Neul et al. 2010. Rett syndrome: revised diagnostic criteria and nomenclature. Ann Neurol . 68, 944...

...but they still have potential targetable sites approximately every 35bp. Researchers are still determining...dimer, cutting in the spacer region between two distinct ZF target sites. (B) A TAL effector nuclease (...available as a kit from Addgene , custom arrays consisting of 12-31 repeats can be assembled and inserted...Toolbox", from Dr. Feng Zhang’s group at the Broad Institute, will be available from Addgene before the end...to mutate a specific gene or your anxiously investigating gene therapy options for a rare disease in humans...constructs for DNA targeting. Cermak T, Doyle EL, Christian M, Wang L, Zhang Y, Schmidt C, Baller JA, Somia...

...must contact your institution’s Bio-Safety office to receive permission and institution-specific instructions... your institution and from the CDC and NIH for work in a BL2 facility. If you have any questions about...Recommended materials C.2 Annealing oligos C.3 Digesting pLKO.1 TRC-Cloning Vector C.4 Ligating and transforming...vector has a 1.9kb stuffer that is released by digestion with AgeI and EcoRI. shRNA oligos are cloned into...selection are continuously being improved. Below are suggestions for target selection. Use an siRNA selection ...targets for your gene. For example, the Whitehead Institute for Biomedical Research hosts an siRNA Selection...Additional design rules can be found at the Broad Institute's TRC shRNA Design Process . To minimize degradation...

... are digesting a large number of plasmids with the same enzyme(s) (for instance, in a diagnostic digest... DNA Ligation Introduction Restriction enzyme digestion takes advantage of naturally occurring enzymes...opens in a new window) NEB . Restriction enzyme digestion is commonly used in molecular cloning techniques...to quickly check the identity of a plasmid by diagnostic digest . Last Upload: Oct. 11th, 2016 Protocol...Pro-Tip If you are conducting a double digest (digesting with two enzymes at the same time), you will need...DNA that you cut depends on your application. A diagnostic digest typically involves ∼500 ng of DNA, while...volume of DNA to be cut. A typical restriction digestion reaction could look like this: 1 µg DNA 1 µL of...

...feedback and suggestions for new antibodies to add to the collection using our Antibody Suggestion Form . Browse...recommended applications based on our in-house testing and data provided by trusted outside labs – with...targeting proteins for neuroscience research. Institute for Protein Innovation Collection : Antibodies...Recommended Applications PI Return to Top Do you have suggestions for other antibodies that should be added to ... represent the official views of the National Institutes of Health. Need Some Help? Browse some of our...

...School Ali Cetin Allen Institute for Brain Science Ben Deverman Broad Institute Katherine Matho Cold Spring...Ali Cetin and Shenqin Yao, Allen Institute Ben Deverman, Broad Institute Katherine Matho, Cold Spring Harbor...Manipulation Meeting Date September 21, 2018 Contact Us Questions? Contact us at [email protected] Sponsor We are...minute presentation, followed by 20 minutes of questions/discussion. The goal of the presentation is to...

...such as titering, purity assessment, sterility testing, and viral genome sequencing.... process: Titering Purity assessment Sterility testing Viral genome sequencing (VGS) Find detailed pricing...Additional steps, including request approval, your institution’s approval of the MTA, and shipping, mean that...order. MTA Approval 1+ business days, varies by requesting organization Viral vector preps remain subject... typically takes 1–4 days, depending on your institution. AAV Production 6–8 weeks Quality is our priority...Addgene does not perform functional or in vivo testing. We recommend that you consult the original paper...

...activation plasmids and screening library CRISPR/Cas9 Synergistic Activation Mediator (SAM) is an engineered protein...the Zhang Lab established Cre-dependent and constitutively expressing Cas9 knockin mice (Platt et al ....sgRNA to a Cre-dependent mouse or sgRNA to a constitutively Cas9-expressing mouse. Described here are AAV...from Jackson Labs: Cre-dependent Cas9 mouse Constitutively active Cas9 mouse Available plasmids are described...plasmid can be digested using SapI, which will reveal sticky ends to enable the rapid ligation of annealed and...

...site) 165508 pOpen-HindIIIR HindIII 165526 pOpen-PstIR PstI 165528 pOpen-SfiIR (cDNA) SfiI (used to create...pOpen-Eco31IB Eco31IB DNA methyltransferase 165536 pOpen-PstIM M.PstI DNA methyltransferase 165538 pOpen-SfiIM M.SfiI...basic and applied fields as diverse as genomics, diagnostics, biodiversity, synthetic biology, DNA origami...pOpen-BovDNTT Bovine DNTT 165555 pOpen-ALPI CIP (calf intestinal phosphatase) Return to top Additional Resources...

...Luciferase-containing plasmids are commonly used to investigate the effect of regulatory elements, such as promoters...constructs available that are driven by a strong constitutive promoter and can be used to monitor transfection...212936 pGL3 Basic Vector Firefly Vector for investigating regions controlling transcription Debrya Groskreutz.../Puro) RapidResponse(TM) Renilla Vector for investigating regions controlling transcription Pete Stecha...212933 pGL4.84(hRlucCP/Puro) Renilla Vector for investigating regions controlling transcription Pete Stecha...Expression Constructs Luciferase driven by a strong constitutive promoter and can be used to monitor transfection...Description Gene/Insert PI Return to Top Do you have suggestions for other plasmids that should be added to this...

...promoters had been identified that responded to stimuli, such as hormones or metal ions; however, these...which can result in unintended system induction. Investigate whether or not your serum is tetracycline-free...replaced with tTA, and Puromycin was replaced with Blasticidin selection. Please NOTE, this vector contains ...(rtTA3) expression vector, CMV promoter, and Blasticidin; can be used to make cell lines Tet-On Advanced...Retroviral; CMV-driven; linked via IRES to EGFP-Blasticidin fusion; pMA2641 has rtTA driven by retroviral...Technology (Link opens in a new window) Do you have suggestions for other plasmids that should be added to this...

...needed. Add stir bar and stir slowly at 4 °C for 1 h, then keep at 4 °C for 3 h without stirring to allow...controller Hazardous waste container pH meter Stir plate Magnetic stir bar Sonicator Ear protection Vortex Reagents...other than PES. AAV particles stick to many other surfaces, but do not stick to PES. Using a PES filter ... FBS media causes less filter clogging during harvesting. The addition of sorbitol has been shown to increase...PEI powder into 100 mL of deionized water. While stirring, slowly add hydrochloric acid until the solution...deionized water and adjust to a final volume of 1000 mL. Stir at RT until fully dissolved. Pro-Tip This step is... challenging due to the high viscosity of PEG. Stirring under medium heat will promote faster dissolution...

...such cases, it may be necessary to customize an existing reagent to suit your needs. Genetic Manipulation...expression vector Cas enzyme promoter can be constitutive (CMV, EF1alpha, CBh) or inducible (Tet-ON); ...genetic knockouts, gRNAs commonly target 5′ constitutively expressed exons, which reduces the chances ...target with no homology elsewhere in the genome. Realistically, a given gRNA target sequence will have partial...protocol associated with the specific plasmid in question (see CRISPR protocols from Addgene depositors ...desired edit, as a large percentage of DSBs are still repaired by NHEJ. How do you determine that your...