...enzyme (among others): caveolin 1, also known as CAV 1. Usually, caveolin 1 promotes the cell life cycle ...caveolin 1, there’s less malignancy because there’s less proliferation. However, the lack of caveolin 1 in ...believes that miRNA may decrease the expression of TAK 1, an enzyme that controls apoptosis and regulates transcription...Agnieszka K., et al. "An absence of stromal caveolin-1 expression predicts early tumor recurrence and poor...to the development and spread of cancer cells. Besides these stromal cell modifications, stromal cells...

...red in the spreadsheet (Figure 2) and are described in Table 1. Some cells have drop-down options for you...Table 1: Guidelines for the data fields in the deposit spreadsheet Data Field Description Plasmid...how to use our deposit spreadsheet to complete Step 1. Steps 2 and 3 are usually very easy — our tech transfer...scientist, you will have the most involvement with Step 1, and, while data entry can be a chore, we’ve made ...Deposit” and “Download Deposit Spreadsheet” (see Figure 1). Please read our “How to Deposit Your Plasmids with...thorough guide to our online data entry process. Figure 1: Begin the deposit process by clicking “Submit Online...lab. Plasmid Type Choose from: Encodes one insert, encodes gRNA/shRNA, or empty backbone. Purpose...

...bacterial mutagenesis or gene targeting Victor de Lorenzo's lab has engineered a modular mini-Tn5 vector...on channelrhodopsins – Chronos & Chrimson Through de novo sequencing of 127 algal transcriptomes, as well...expression. Brown et al., Genesis 2014 Jan;52(1):39-48. APEX2 for proteomic mapping and electron...engineering cells that lack release factor one (RF-1; Bacterial strain EcAR7) and minimizing the set of... of human mitogen-activated ERK activating kinase 1 (MEK1) with either one or two phosphoserine residues...comprised of three sets of cloning vectors (Level 0, 1, or 2) which can be utilized in three successive assembly... vectors are directionally assembled into a Level 1 vector creating a single transcriptional unit (Ex:...

...and placed in a buffer. Antibody Structure Figure 1: Structure of an antibody A standard antibody is made... together to form a “Y” shape, as shown in Figure 1. The two arms of the Y structure are responsible for... is described in more detail in the Multiplex Assays section below. The following table describes the ... Science Guides Antibody Guide Antibody Guide View Addgene's Antibody...of B cells isolated from animals immunized as described above are used to create hybridomas, which produce...each antibody has two binding sites. Isotype Description Configuration and Valency Subclasses IgA Prevalent... used to quantify relative amounts of protein. Includes: Western blot ELISA Capture - used to isolate ...

... handed out at the event). Program 0 - 1:24 - Intro 1:24 - 29:31 - Reproducibility Overview - Jeffrey... 49:45 0 1:18:45 - Reagent Development - Steven C. Almo, Institute for Protein Innovation  1:18:45 - 2...reproducibility As Jeffrey Flier points out in his talk (1:24 - 29:31), there are a variety of ways of defining...enterprise towards greater reproducibility. Cause 1: Inability to repeat previously performed experiments...the Institute for Protein Innovation (IPI) (49:45 - 1:18:45), there are also initiatives to create more ...found. Consider the hypothetical situation in Figure 1 where many scientists are performing similar experiments...experimental designs and we need to be transparent about the reasons we alter these designs if we do so...

...applications. LuxSit-i : An artificial luciferase desined de novo using deep-learning approaches. Split-Luciferase... Eric Campeau 21474 pLenti CMV V5-LUC Blast (w567-1) Firefly CMV Lentiviral expression of firefly luciferase...to that of the fluorophore. Conformationally destabilized Firefly luciferase mutants : A set of three ...three sensors that are increasingly structurally destabilized versions of Firefly luciferase which serves ...dimerization , endosomal escape of cell-penetrating peptides , and more. nLuc/cLuc for split Firefly Luciferase...mammalian systems. ID Plasmid Luciferase Type(s) Description PI 16539 pBV-Luc Firefly Insertion of 5' promoter...service icon . ID Plasmid Luciferase Type Promoter Description PI 60226 AAV:ITR-U6-sgRNA(backbone)-pEFS-Rluc...

...estimated to affect anywhere from 1 in 6,000 to 1 in 19,000 live births (1).  While disruption of the smn1... funding opportunities. References 1. Jones, C., et al. "PP09. 1–2352: Systematic review of incidence...rare diseases. These underrepresented individuals deserve a voice. Check back with Addgene often to learn...

...deletions, point mutations PCR Reactions 1 1 1 per fragment 1 per fragment In vitro Assembly No No Yes...methods for site directed mutagenesis   References 1. Trehan, Ashutosh, et al. (2016). “REPLACR-mutagenesis...discussed methods. As described above, you design two primers, each containing the desired mutations, to amplify...make a point mutation, you design two oligonucleotide primers with the desired mutation, then use those ... figure above shows guidelines for primer design; a desired insertion should be present in both primers...vitro recombination. Across all modifications described in Trehan et al., REPLACR-mutagenesis’ median ...

...sensing glutamate using both 1-photon and 2-photon imaging.   Figure 1: 1P and 2P excitation and ...hSynap: Human Synapsin-1 promoter. Good for neuronal expression. hSynap-FLEX: Synapsin-1 promoter, FLEXed for... v857. Image adapted from Aggarwal et al., Figure 1   Increased versatility through two different...expression in Cre-expressing cells. hSynap-FLP: Synapsin-1 promoter, FLP-dependent expression GFAP: Glial fibrillary...characterization of the new variants is available here. Upgrades to iGluSnFR3 To improve upon the SF-iGluSnFR, ...

...template (Fig. 1, Step 1). There is no need for PCR or cloning, as digestion of the destination vector with...selectable marker, and connector to be released (Fig. 1, Step 2-3). Since donor plasmids of the same type ...correctly assembles the final targeting vector (Fig. 1, Step 4), and co-injection of the targeting vector...human cells. To use CRISPaint, users need 3 vectors:  1) a gRNA vector that targets the gene of interest, ... Tagging with Short Homology Donors.” Cell Rep.15(1) (2016):210-8. PubMed PMID: 27052166. Kunzelmann,...SapTrap, the user first designs either oligos or synthetic DNA for the desired gRNA target sequence, as...specified by a guide RNA (gRNA) sequence. Users can design gRNAs to induce the break at a specific genomic...

...bidirectional control of neuronal activity. Table 1. Descriptions of DREADDs and their activity in neurons DREADD...out our Chemogenetics Plasmid Collection . Figure 1: General overview of a chemogenetics experiment. Early... while PSAMs and LMOs have an effect for only 0.5–1 hours after delivery. Chemogenetic ligand. The chemogenetic...a new window) Guettier JM, Gautam D, Scarselli M, de Azua IR, Li JH, et al. (2009). A chemical-genetic...less than ideal. DREADDs Designer Receptors Exclusively Activated by Designer Drugs (DREADDs), like RASSLs...Relationship Studies for Designer Receptors Exclusively Activated by Designer Drugs. ACS Chem Neurosci...Wess J (2012). Design and functional characterization of a novel, arrestin-biased designer G protein-coupled...

...pLenti CMV Puro DEST (w118-1) or the doxycycline-inducible pLIX_403. The chosen attR destination vector will... our desired constructs: lentiviral expression of the human KRAS gene in mammalian cells. STEP 1: Generate...recombination sites (attL-left- and attR-right-, Figure 1). Under certain conditions, the attL and attR sites...regeneration of attP and attB sites.   Figure 1: Lambda phage integration and excision reactions. ...the generation of the expression construct in only 1 day, as opposed to 2+ days with traditional restriction... Find Gateway cloning plasmids here! References 1. Chee JY, Chin CF (2015) Gateway Cloning Technology...clone in their desired DNA sequences. Gateway technology relies on the two reactions described below: The ...

...reactions. (Need a refresher? The five rules are: (1) don’t use the same overhang twice; (2) avoid palindromes...Gate) pot (Pryor et al., 2022).     Figure 1: Workflow for high complexity Golden Gate Assemblies...different enzymes and cycling conditions (see Table 1).   Enzyme Temperature Incubation time/cycling...cycling Overhang length T4 DNA ligase 25 °C 1 hour 3-base or 4-base T4 DNA ligase 25 °C ...hours 3-base or 4-base T4 DNA ligase 37 °C 1 hour 3-base or 4-base T4 DNA ligase 37 °C ...Ligase MM 37-16 °C cycling 3-base Table 1: Enzyme and cycling conditions available in DAD tools...rules of thumb for designing your overhang sets. But are those rules the best way to design GGA overhang sets...

... that includes the aforementioned internal control and is consistent with the result in Figure 1 could...Gene X using shRNA expressed from a plasmid   Figure 1: Expression Level of Gene X The results shown above...Referring back to the experiment associated with Figure 1, the independent variable is the shRNA. Therefore ...the protein.   In analysis of the result in Figure 1, an internal control, such as the GFP-expressing Plasmid...plasmid.  Let us now revisit our experiment. In Figure 1, it appeared as though the shRNA did not knock down...scientific inquiry and investigation. References: 1. The problem of pseudoreplication in neuroscientific...and should be designed accordingly. If you are trying activate a gene, you should design a control that...

...to the most colonies after transformation. When a 1:1 vector:insert ratio is used in the DpnI digestion... get tips for applying it in your own lab. Table 1: Comparison Between Standard Restriction Cloning and...Connecticut interested in protein science.     References: 1. Celie, Patrick HN, Annabel HA Parret, and Anastassis...PCR cloning method." BMC biotechnology 11.1 (2011): 1. PubMed PMID: 21992524. PubMed Central PMCID: PMC3207894.... Traditional restriction cloning is one of the oldest of these techniques and is a multi-step process...

..., they used 1-2 CRISPR crRNAs targeting sequences found only in certain bacteria (Figure 1). Citorik et...from 50% to 11% in just 24 hours.   Figure 1: Single-phage strategies for bacterial CRISPR targeting...genome, and it carries CRISPR machinery targeting 1) the given resistance gene and 2) a second lytic phage...the fight against bacterial superbugs. References 1. Citorik, Robert J., Mimee, Mark, and Timothy K. Lu...Targeted Bacterial Population Editing.” Cell Syst. 1(3):187-196. (2015). PubMed: PMID 26973885. Additional...beta-lactam resistant population to the antibiotic, despite this resistance gene being encoded by a high-copy...currently undergoing a renaissance. Yosef et al. designed a two-phage CRISPR system to resensitize antibiotic-resistant...

...the steps needed for TOPO cloning: 1. Create Your PCR Product: Design standard primers (no need to add ... strand and a tyrosyl residue of topoisomerase I (1). If a 5′ hydroxyl group from a different DNA strand...mind that Taq polymerase has an error rate of about 1 in 3,500 bases. Typically polymerases with proofreading...enzyme and Taq, with Taq used in an excess ratio of 10:1. Gel purify your PCR product and incubate it with ...you to see colonies within 8 hours. References: 1. Shuman S. "Recombination mediated by vaccinia virus...

...need a job. Start now. Informational interviews are 1:1 discussions with someone who has interesting experiences...conversation builds a relationship for the future and the 1:1 format is good for everyone including those that ...out-of-the-box ideas that might jump start your learning. 1. Create a peer mentoring group We don’t all get the...harder to build relationships when you are more desperate or needy so you can’t wait until you imminently...

...family including human immunodeficiency virus type-1 (HIV-1), can infect both dividing and non-dividing cells...internal promoter, and the desired gene (transfer vector plasmid), 2) the HIV-1 genome containing all viral...to test several protocols that have been described (protocol 1, protocol 2, protocol 3) to find the one...plasmids.  References 1. Miller, A. DUSTY, and C. A. R. O. L. Buttimore. "Redesign of retrovirus packaging...have been devoted to develop efficient and safe HIV-1-derived lentiviral vectors due to their ability to...lentiviral vectors consisted of three plasmids encoding 1) the lentiviral vector genome which was composed of...generation because this vector requires only three HIV-1 genes (gag, pol, and rev) for production. Third generation...

...protocols. A typical ExM protocol (Fig. 1) follows this structure: Step 1: fix (and maybe label) your sample...microscopy. Nat Biotechnol 1–12. https://doi.org/10.1038/s41587-022-01546-1  Mikhaylova M, Cloin BMC, ...just adding water.)  Step 5: Image   Figure 1: The ExM process allows you to physically magnify ...16:71–74. https://doi.org/10.1038/s41592-018-0238-1  Klimas A, Gallagher BR, Wijesekara P, et al (2023...Microscopy. What is Expansion Microscopy?  ExM provides a straightforward answer to the space problem....improve label density (Mikhaylova, et al. 2015).  Despite these limitations, ExM is a valuable tool, especially...Bournonville L, et al (2021) Expansion microscopy provides new insights into the cytoskeleton of malaria ...