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...prep. With this in mind, we recommend that users titrate their AAV vectors to find the optimal dose that...Quality Control Resources on Addgene.org Find Ready-to-Use AAV Find Ready-to-Use Lentivirus Browse Viral ...used to repeating experiments to confirm their findings. But no scientist wants to repeat an experiment...step process using Geneious software: First, the individual sequencing reads (~150 bp each) are aligned to... reviewed. As mentioned earlier, it is common to find DNA from the packaging cell genome, bacterial genomes...our pipeline (see Figure 2, sample 2). When we blindly analyzed AAV-68544, we were easily able to identify...Recombinase-dependent sequences may be subject to recombinase-independent recombination during plasmid growth in bacteria...

...transcription factor binding site. GAL4 expressed in the fly binds to the UAS binding motif, driving downstream...cells in a population, it’s easier to visualize individual/non-overlapping cells. While transgenic animals...require heat-shocking the system or using chemical inducers of gene expression (del Valle Rodríguez et al....controlled by the upstream recombination outcome. Find SPARC plasmids at Addgene To more precisely control...intermediate (d), and sparse (e) labeling of the calcium indicator, GCaMP6f, in the Drosophila optic lobe. Image ...organisms by replacing the transcription factor binding site and effector in the construct. In Drosophila...dendrites of T5 neurons by placing the calcium indicator GCaMP6f in the effector position, as well as to...

... of the antibody has an antigen binding site for a total of 2 binding sites per antibody. C) IgG’s are...Advantages of Nanobodies How to Use the Nanobody Toolbox Find Nanobody Plasmids Structural comparison As the...Antibodies One of the main reasons antibodies are great indirect immunodetection tools is their strong signal strength...occur: A) Multiple polyclonal secondary IgGs can bind to different epitopes of a primary antibody. B) ...displacement 2° incubation can be skipped by pre-binding to 1° Monovalent Monoclonal Only anti- rabbit...IgG subclasses. Instead, you can pre-incubate individual primary antibodies with secondary nanobodies ... with three IgG1 antibodies. Antibodies were individually incubated with a secondary nanobody conjugated...

...100nt window upstream of the transcription start site (TSS), while for CRISPRi, a ~100nt window downstream...Basics with Our CRISPR Guide Find CRISPR Plasmids for Your Research Find CRISPR Pooled Libraries for Your...important considerations for designing your gRNAs. For indels, it's not so important what location in the gene...joining (NHEJ) often leads to the generation of indels and thus frameshifts that disrupt the protein-coding...consuming process and should not be undertaken lightly! Indeed, truly achieving the gold standard requires not...editors, the intended edit must be in a 5 - 10 nt window relative to the PAM, and bystander edits are possible...possible if there is another target C or A in the window. Another technology, prime editing (reviewed in...

... of well characterized inducible promoters. Far removed from the bio-industry’s vision of ‘biofactories...multiple scRNAs all individually utilize the same dCas9 protein. By using an inducible promoter to modulate...that can fold into a secondary structure that can bind specific proteins via aptamer domains. Combined ...Combined with dCas9, this new scaffold RNA (scRNA) will bind to a PAM-adjacent target nucleotide sequence then...transcriptional regulator or the number of designed scaffold binding sites. With hundreds of characterized aptamer domains...relies on base pairing, it has promise to be host-independent, beyond the oft published metabolic engineering...recognize guide RNA but with a different dCas9 binding module, alternative sets of transcriptional programs...

...our Hot Plasmids articles.  Here's what you'll find in this post: Anti-V5 and other new epitope tag...Anderson et al., 2024 under CC BY license.    Find Anti-V5 [IPI-SV5-Pk1] Antibody here!   Anderson...studies of protein ubiquitination and degradation.   Find the CRBNmidi plasmid here!   Kroupova, A. et al...and prolonged imaging with minimal background.   Find these C. elegans mScarlet plasmids here!   Cao ...resistance interfering with selection. Using an inducible Cas9 expression and removing the sgRNA plasmid...site, which results in either bacterial death or indels after DNA repair. B) M. abscessus is first transformed...resistance selection marker HygR. The CRISPR system is induced to cause disruption of the target gene. Lastly,...

...another edition of Hot Plasmids!  Here's what you'll find in this post:  New compact, efficient prime editors...templates and significantly reduced undesired edits (indels, etc.), while PE6c and PE6d were most efficient...Pandey et al., 2023 under CC-BY 4.0 License. Find PE6 plasmids here!  Doman, J.L., Pandey, S., et ...experimental therapies and medical applications.  Find DIRECTED plasmids here! Strebinger, D., et al. (...in neurons, in pulse-chase experiments, and more. Find LOV-Turbo plasmids here! Next, Qin and Cheah et ...incorporated LOV-Turbo into TransitID experiments.  Find TransitID plasmids here! These two developments ...labeled. Using lentiviral vectors to stably or inducibly express tagged iPAK4 from different promoters,...

...meeting people with different mindsets and approaches can truly broaden your mind. When I first began collaborating...practical tools like alumni networks. I find it useful to remind myself to check in regularly with collaborators...people at Addgene) had to take the initiative to find a suitable partner, connect the relevant teams, ...types of events can be very tough. First I needed to find the right contacts at the companies and then convince...the perspective of my potential collaborators in mind. Being at a university allows you to approach people...sponsors for a training weekend for a sports club, find speakers for a meetup or recruit volunteers for ...: maintaining these relationships. Personally, I find this to be the most challenging and time-intensive...

...academia or industry. At BosLab, we identify ourselves as part of a network of independent Do-it-Yourself...building and you will find a bike kitchen on the first floor and upstairs you will find our fully-equipped...addition to maintaining a laboratory to support both individual and community science projects, we offer classes...equipment. It was humble in comparison to the labs you’d find in Kendall square, but we saw the potential in the...club events and soon realized that it was easy to find people who were interested. For example, our journal... our members start out with personal projects in mind. Thus, to encourage more involvement and provide...creations that may not be pursued in an academic or industry lab. Mrina Nikrad, Ph.D., is an expert in microbial...

...variable fragment (scFv), grafting the binding region of a FLAG-binding scFv onto a well-folded scFv scaffold...SDS-Page gels. A: individually expressed proteins run in separate lanes. B: individually expressed and coexpressed...deposits and we hope they'll make it easier for you to find and use the plasmids you need. If you'd ever like...deposit please sign up here.  Here's what you'll find in this post: Anti-FLAG Frankenbodies for modular...scFv scaffold. (b) Schematic of anti-FLAG-FB-GFP binding to FLAG-tag H2B for nuclear localization. (c) Applications...selection rules, and reducing recombination by finding acceptable variations in DR sequences. Figure...60, 80 and 100 kD proteins in E. coli, either individually (~20,000 lanes/50 mLs) or through two polycistronic...

...contains a 4-nucleotide Rep binding element (RBE) that serves as a binding site for Rep78 and Rep68 to...rep (replication) and cap (capsid) proteins, which bind to the ITRs and initiate replication. ITRs are cis-acting... 1). Everything else outside the ITRs gets left behind.    Figure 1: Recombinant AAV vector with ITRs...the gene of interest. A single ITR consists of palindromic arms (A-A’, B-B’, and C-C’) that give the feature...-shape. The arrangement of the B-B’ and C-C’ palindromic sequences determines the orientation of the ITR...and flop orientation. The ITR contains three palindromic sequences (A-A’, B-B’, and C-C’ regions) and ...and forms a T-shaped structure. The Rep binding element (RBE) and terminal resolution site (trs) are noted...

...comprise a DNA binding domain and an endonuclease domain. What’s distinctive is that the DNA-binding domain is...could potentially sequence a large number of individuals and find several mutations in the same gene or in... is modifiable and can be programmed to bind to essentially any DNA sequence desired. This technology ...prefabricated pieces that allow you to build a DNA-binding domain to recognize any desired 15 base pair sequence...electroporation. All you do to create a protein that binds those 15 base pairs is take four plasmids out of...about the history. How had investigators done this kind of work before? Musunuru: In the "olden days," not...expertise to create zinc finger nucleases that will bind desired sequences. TALENs are much more reliable...

... our CRISPR Protocols Find gRNA Design Tools Read other CRISPR blog posts Find CRISPR Plasmids at Addgene...screening success? Before you start analyzing individual clones, it’s wise to quickly assess whether a...haystack before going on to generate and screen individual clones. If a clonal line isn’t your end goal,...popular screening method for knockouts is Tracking of Indels by Decomposition (TIDE) (Brinkman et al., NAR)....and deletions identified within a given genomic window, along with the estimated editing frequency of ... can determine if it is worthwhile to isolate individual clones from that population. You can additionally... 25% are +/+ for your gene of interest. Thus, to find a null clone, you should screen, at the very least...

...your timeline Start with your thesis in mind. Imagine the kind of data your dream discovery will need,...books.  To find personnel salary ranges, go to online job information sites (like, indeed.com, or salary.com...palms. Heart racing. Cotton mouth.The candidate may find it difficult to answer and these are all symptoms...how long it will take to publish based on these findings. Most often we suggest estimating the time it ...experiments, start defining keywords you’ll want to find in candidates’ resumes or CVs; “vivarium”, “weaning...salary.com). Search industry and academic job titles in the regions where the host institute is located. ... your work requires specialized equipment, try finding institutes that offer shared equipment that you...

...you should be asking. What kind of control? On, off, tunable, and/or inducible? How long do you need control...expression for good, the Cre-Lox system offers inducible yet permanent gene excision. Essentially, LoxP...strand break. Transient introduction of CRISPRi or inducible expression (more on this below) will both achieve...tissue-specific targeting. Looking to permanently induce expression? Check out our Retro expression vector...and repressor mechanisms Want to transiently induce expression? Check out Addgene's Lentiviral dCas9...Addition of tetracycline or one of its analogs will induce these transcriptional changes, and expression is...relevant proteins with a degron tag can provide inducible protein degradation. These systems work by tagging...

...details or permission to distribute, or Addgene finds your journal publication describing the plasmids... inserted into it. You will have the option to indicate separate promoters, tags, or cloning information...strain(s) through Addgene. Description – please indicate the gene cloned into your plasmid, any tags or...information. Enter plasmid information During the remainder of the process, you will enter plasmid data in...this will make it easier for other scientists to find information related to your plasmid. It is optional...insert using standard mutation notation. Please indicate the amino acid change as well as any functional...etc) on your insert. Use the drop-down menu to indicate whether the tag is on the N or C terminus and ...

...plasmids, with many available to scientists in industry. Since March 2020, we have received over 13,000...SARS-CoV-2 virus depends on its spike protein to bind ACE2, the host cellular receptor. To more easily... of antibodies from 19 COVID-19 convalescent individuals (Tong et al., 2021). Another version of the spike...determine how effective sera from vaccinated individuals are at neutralizing the different SARS-CoV-2 ...selection. For more information on these plasmids and a behind-the-scenes glimpse of their work, head over to ...glad to join these efforts and help academic and industry scientists through plasmid sharing and more. As...Ludes-Meyers J, Nguyen AW, Park J, Wang N, Amengor D, Lavinder JJ, Ippolito GC, Maynard JA, Finkelstein IJ, McLellan...

...common drug used to induce the expression of Cre is tamoxifen. In tamoxifen-inducible Cre (Cre-ER) systems..., knockout mice, and mice with conditional or inducible gene expression. Transgenic and knockin mice As...into a female animal for gestation. Nucleases that induce double-stranded DNA breaks, such as ZFN and TALEN...targeted to specific regions of the genome, and induce non-homologous repair at the site of DNA damage... identify the ones with the desired genotype. Inducible and conditional systems Conditional knockouts ...DNA from one region of the genome to another.  Inducible gene expression Another type of genetically engineered...systems, Cre is fused to the ligand binding domain of estrogen receptor alpha (Esr1). Mice are fed or injected...

...Let’s start by examining individual parts and transcriptional units. Individual parts are defined DNA sequences...multi-part DNA assembly. Simply put, it assembles individual DNA components into larger units or circuits ...particular cloning technique. Though rarer, you can find MoClo that relies on Gibson and other non-Golden...be broken down into three hierarchical levels: individual DNA parts, transcriptional units, and multigene... sequences with specific functions. Most individual parts have characterized activities with predictable...While a gene is the most obvious example of an individual part, this category also includes promoters, ..., UTRs, terminators, etc. Once selected, individual units can be combined into… Transcription units (TUs...

...contain a variety of clones and thus the potential to bind many different sites on the target protein they ...fraction of the antibody molecules will recognize and bind to your primary antibody. Manning et al. tested ...present in a broadly reactive secondary antibody will bind to a monoclonal primary antibody (A). To boost signal...increase the chance of primary/secondary antibody binding and therefore the signal strength (Fig. 1). Monoclonal...with higher affinities, like those from a rabbit, bind a greater amount of antigen in a shorter period ...better choice for assays that require high affinity binding, such as immunoprecipitations. Your sample species...anti-mouse secondary antibodies used for detection bind to non-target immunoglobulins naturally present ...