...networks, and specific neuronal populations to consider. To tease apart how different neurons impact a...experimental approach you choose, it’s essential to consider the technical limitations of each approach and...

...conducted in Professor Cepko's lab and to get some insight into her management and mentoring styles. If you'd...

...better and make scientists’ lives easier. Why transition to Addgene? So why did I join Addgene? Was I ...easier. I’m hoping my story demonstrates that transitioning from academia doesn’t have to mean abandoning...graduate training. PhDs have many roles to play inside, on the outskirts, and entirely outside of academia...

...Takeharu Nagai Tension TS module, Vinculin tension sensors Measuring mechanical tension across vinculin...7303):263-6. Martin Schwartz Tension Talin tension sensors Talin tension sensor reveals novel features...F40-based tension sensor modules Grashoff Lab Tension Sensor Plasmids Carsten Grashoff Tension tCRMod tension... tension sensors Tunable molecular tension sensors reveal extension-based control of vinculin loading.... cAMPFIRE cAMP biosensors with improved sensitivity Sensitive genetically encoded sensors for population...highly-sensitive genetically encoded temperature indicator exploiting a temperature-responsive elastin-like...transmission and mechanosensitivity. J Cell Biol. 2016 May 9;213(3):371-83. Martin Schwartz Tension HP35-, FL...

...amino acid linker ACA Heterotrimeric construct consisting of Amber-5aa linker-Cerulean-6aa linker-Amber...linker-Amber ACV Heterotrimeric construct consisting of Amber-5aa linker-Cerulean-6aa linker-Venus VCA Heterotrimeric...Heterotrimeric construct consisting of Venus-5aa linker-Cerulean-6aa linker-Amber VCV Heterotrimeric construct...construct consisting of Venus-5aa linker-Cerulean-6aa linker-Venus ACAV Heterotetrameric construct consisting... linker-Venus ACVA Heterotetrameric construct consisting of Amber-5aa linker-Cerulean-5aa linker-Venus... linker-Amber VCAA Heterotetrameric construct consisting of Venus-5aa linker-Cerulean-5aa linker-Amber... linker-Amber VCVV Heterotetrameric construct consisting of Venus-5aa linker-Cerulean-5aa linker-Venus...

...Figure 1. The two arms of the Y structure are responsible for binding the antigen, and are called the antigen...isotype of the antibody. B cells, the immune cell responsible for producing antibodies, can change the constant...binding site. In B cells, the genetic region responsible for the variable region goes through a randomized...valency of 2 IgE Protects against parasites and is responsible for driving allergic reactions such as anaphylactic...requirements, monoclonal antibodies are typically more expensive than polyclonal antibodies. Environmental factors... Minibodies are engineered antibody fragments consisting of two scFvs bound to a CH3 domain. Fab fragments... protein. Fluorophores Fluorophores are light-sensitive molecules which emit a specific wavelength of ...

...(Quorum sensing molecule) Cynthia Collins Contains luciferase inducible by the quorum sensing molecule...transcription factor responsive to 3OC6HSL. EsaR derivatives found herein have different sensitivities to 3OC6HSL...expression of Cas9 nuclease gRNA. For a more comprehensive list of our bacterial CRISPR plasmids, see here...inducing double stranded breaks. For a more comprehensive list of our bacterial CRISPR plasmids, see here...bacterial gene disruption or knockdown. For a more comprehensive list of our bacterial CRISPR plasmids, see here...Protein Purification Sometimes looking at a protein inside cells just isn’t enough to get all the information...3OC6HSL. The EsaR-responsive promoter can be found in pCS-PesaRlux controlling luciferase expression. pCW-LIC...

... proteins consists of Type I, Type II, and Type III interferons. They trigger the defensive responses ..., WBCQ1 DEFA1 defensin, alpha 1 DEF1, DEFA2, HNP-1, HP-1, MGC138393, MRS DEFA3 defensin, alpha 3, neutrophil-specific..., HP-3 DEFA5 defensin, alpha 5, Paneth cell-specific DEF5, HD-5, MGC129728 DEFB1 defensin, beta 1 BD1,...MGC51822 DEFB103A defensin, beta 103A DEFB103, DEFB3, HBD-3, HBD3, HBP-3, HBP3 DEFB104A defensin, beta 104A ...

...erythroleukemia (MEL) cells, a suspension cell line. The culture medium in all steps consists of DMEM supplemented...eukaryotic genome engineering. CRISPR/Cas9 is an inexpensive, facile, and efficient genome editing tool that...off-target effects). Ensure that the guide sequences consist of a 20-mer (“protospacer sequence”) upstream of..., resolve agarose gel to determine if size is consistent with the presence or absence of deletion. For...L-glutamine, which is used for MEL cells. However, transient transfection of CRISPR/Cas9 plasmids may be successfully...strategies for each cell type. While MEL cells are suspension cells, instructions for adherent cells have ...validation (see step 6 ). NOTE: This protocol is for suspension cells. Adherent cells can either grow as individual...

...apply to using CRISPR in other organisms. First, consider the genetic manipulation that is necessary to ...Manipulation Application Cas9 gRNA Additional Considerations Knockout Permanently disrupt gene function ...ribonucleoprotein (RNP) complexes are advantageous due to the transient Cas9 expression. The table below summarizes the...details, see Plasmids 101: Mammalian Vectors ) Transient or stable expression of Cas9 and/or gRNA in a ...limit (only compatible with smaller Cas enzymes) Transient or stable expression of SaCas9 and/or gRNA Infects...cells through microinjection or electroporation Transient expression of CRISPR components Expression decreases...which is delivered to cells using cationic lipids Transient expression of CRISPR components Short window of...

...receptor for SARS-CoV-2 and SARS-CoV is hACE2, an angiotensin receptor. Another key step is the priming of ...to identify antibody escape mutants and for comprehensive mapping. Some of the following plasmids are ...the links and references below: ACE2 - (Human Angiotensin Converting Enzyme 2) is the host cell receptor...SHERLOCK and DETECTR - Simple, portable, and inexpensive platforms to reliably detect nucleic acids at...and optimize the method termed Specific High Sensitivity Enzymatic Reporter UnLOCKING (SHERLOCK and SHERLOCKv2...available from Xiaokun Shu's lab. A rapid, highly sensitive and open-access SARS-CoV-2 detection assay for... Library of Spike protein RBD mutations for comprehensive mapping (Jesse Bloom). SARS-CoV-2 Spike Receptor...

...MCP tagged plasmids for visualization of proteins inside cells....ACP-, and MCP-tags for visualization of proteins inside living or fixed cells. The plasmids associated ...visit the New England Biolabs website. For a comprehensive comparison to GFP, please refer to NEB's comparison...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...pMCP-tag (m) Vector ACP/MCP-tag Empty backbone for transient mammalian expression 101126 pACP-tag (m)- 2 Vector...Vector ACP/MCP-tag Empty backbone for stable and transient mammalian expression 101132 pMCP-GPI Control Plasmid...

...for your CRISPR genome engineering experiments, consider that wildtype Cas9 with optimized chimeric gRNA...concentration of Cas9 and guide RNA (gRNA). These considerations only matter if the PAM sequence is immediately...to generate off-target DSBs; there needs to be extensive homology of the protospacer followed by PAM. Click...vector design guidelines? A few notes below are considerations for designing HR donor. Generally if off-target...10bp away. For small (<50bp) changes, you should consider using ssDNA oligo as donor template, it's usually...selected/screened? Before starting your experiment, consider co-transfecting with GFP. This allows you to sort...

...Viral vector production Density centrifugation purification Addgene’s comprehensive quality control process... amplify DNA, produce the prep, and perform comprehensive quality control processes to make sure it is...vector experience, from methods to troubleshooting insights, you make research more reproducible for scientists... that complement published data, capturing the insights that often go unshared. Help Center Check out ...

...fusing the anti-CRISPR protein AcrIIA4 with the photosensitive LOV2 domain . Several labs have also controlled...There are two main types of base edits: Base transitions - edit to same classification of nucleotide base...pyrimidine-to-pyrimidine), which catalyze base transitions. Cytosine base editors are created by fusing ...integrated along with the sequence, so the edit is not considered “scarless”. Figure 7: Overview of CRISPR transposases... target gene function. It has also been used extensively to screen for novel genes that regulate known...simplest dCas9-based activators and repressors consist of dCas9 fused directly to a single transcriptional...silencing across much of the human genome following transient expression of the dCas9 fusion, with the epigenetic...

...backbone to use, you have many elements to consider. Here is a guide to Addgene's empty vector backbones...Mammalian CMV, SV40, EF1a, CAG pSG5L Flag HA - Transient expression pWPXL - Lentiviral ...expression pBI-MCS-EGFP - Tet-inducible Transient expression - Gradia Lab plasmids...to Top Viral Expression and Packaging Although transient expression is sufficient for some experiments,...and nondividing cells Addgene has an extensive collection of plasmids for packaging...

...known half-life (24 hours), and is compararably inexpensive. The induction profiles for other tetracycline...β-globin poly A) & EGFP from a bidirectional tet-responsive promoter (Pbi); Pbi contains a TRE between two...gene of interest & GFP from a bidirectional tet-responsive promoter (Pbi); Pbi contains a TRE between two...expression in mammalian cells by tetracycline-responsive promoters. Gossen M and Bujard H. PNAS . 1992...2006 Oct;13(19):1382-90. PubMed . Improved Tet-responsive promoters with minimized background expression...

...Browse the Zhang lab’s comprehensive human transcription factor collection, available at Addgene. The...Plasmids MORF Pooled Library The MORF library is a comprehensive human transcription factor collection that contains...Pooled Library The MORF pooled library is a comprehensive collection of human transcription factor open...

...as logic gates and higher level gene networks. Sensing and Signaling Browse plasmids related to intercellular...intercellular signaling and environmental sensing. Strains Browse Addgene's collection of engineered bacterial...Yeast-Mammalian Circuit Transfer C. Collins Lab Quorum Sensing Ellis Lab GeneGuard Endy Lab Logic Gates , BIOFAB...

...and stained with Coomassie blue. Protein band intensities are measured using ImageJ software and a standard...standard curve. Using ImageJ software, the band intensities of both the antibody and impurities are measured...anti-GFP antibody), the target antigen is first transiently expressed in mammalian cells. To learn which ...