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  1. Rewiring Metabolic Circuitry with CRISPR RNA Scaffolds [Video]

    Type
    Blog Post
    Published
    April 7, 2015, 4:21 p.m.
    ... 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...

  2. Transferable Skills Guide: External Collaborations

    Type
    Blog Post
    Published
    April 19, 2018, 1:25 p.m.
    ...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...

  3. Hot Plasmids: Fall 2024

    Type
    Blog Post
    Published
    Nov. 21, 2024, 1:30 p.m.
    ...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,...

  4. Hot Plasmids - November 2023

    Type
    Blog Post
    Published
    Nov. 14, 2023, 3:35 a.m.
    ...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,...

  5. Viral Vectors 101: Inverted Terminal Repeats

    Type
    Blog Post
    Published
    Oct. 19, 2023, 1:15 p.m.
    ...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...

  6. Cultivating Community Science at BosLab

    Type
    Blog Post
    Published
    Nov. 2, 2017, 1:35 p.m.
    ...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...

  7. Hot Plasmids - February 2022

    Type
    Blog Post
    Published
    Feb. 22, 2022, 2:15 p.m.
    ...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...

  8. Kiran Musunuru on the Newest TALEN Genome-Editing System

    Type
    Blog Post
    Published
    Dec. 10, 2013, 3:13 p.m.
    ...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...

  9. CRISPR 101: Validating Your Genome Edit

    Type
    Blog Post
    Published
    Nov. 3, 2022, 12:15 p.m.
    ... 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...

  10. How to Negotiate a Successful Lab Start Up Budget

    Type
    Blog Post
    Published
    Sept. 1, 2015, 2:30 p.m.
    ...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...

  11. Plasmids 101: The protein expression toolbox

    Type
    Blog Post
    Published
    Feb. 16, 2023, 7:05 p.m.
    ...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...

  12. Mouse Modeling, Part 1: Genetically Engineered Mice

    Type
    Blog Post
    Published
    July 11, 2019, 1:26 p.m.
    ...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...

  13. A Look Back at One Year of Plasmid Sharing for COVID-19 Research

    Type
    Blog Post
    Published
    July 12, 2021, 1:15 p.m.
    ...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...

  14. Plasmids 101: Modular Cloning

    Type
    Blog Post
    Published
    March 12, 2024, 1:15 p.m.
    ...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...

  15. Antibodies 101: Choosing the Right Isotype

    Type
    Blog Post
    Published
    April 6, 2023, 3 p.m.
    ...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 ...

  16. Plasmids 101: Degron Tags

    Type
    Blog Post
    Published
    March 14, 2024, 1:15 p.m.
    ...either the N or C terminal of a protein and can inducibly facilitate the degradation of the tagged protein...mechanisms of control. Protein degradation can be induced by the addition of a small molecule, and protein...degron tags can do more than just degrade! Read on to find out more. Common degron tags When it comes to degrons...novel degrader, dTAG13, is a small molecule which induces dimerization of the FKPB12F36V fusion protein with... also be handy!   Fig 1. dTag molecules induce dimerization of an FKBP12F36V fused target protein-of-interest... can be added to media, buffer, etc., which will bind and light up a tagged protein for imaging. Given...ligases to specific proteins by simultaneously binding both their target protein and an E3 ligase through...

  17. Truncated gRNAs for Regulating Gene Expression

    Type
    Blog Post
    Published
    Jan. 10, 2017, 3:37 p.m.
    ...of making targeted genetic edits. Cas9 is a DNA binding protein that can be directed to virtually any genetic..., each of which cleaves one strand of DNA upon binding the protospacer. The resulting double strand break...respectively. When complexed with a truncated gRNA, Cas9 binds the target site, but does not cut either strand ...fusion proteins retain the ability to recognize and bind to DNA; when complexed with a gRNA that directs ...of attempting to simultaneously and selectively induce both cutting and regulation of expression at different...of complementarity to a target site, Cas9’s DNA binding capability remains intact while its nucleolytic...transcriptional start site (TSS) of a gene of interest, it induces potent gene expression with no genetic alterations...

  18. Sensing Neuronal Dopamine

    Type
    Blog Post
    Published
    July 30, 2019, 12:59 p.m.
    ...Learn about Addgene's viral service Find viral protocols and videos Find biosensor AAVs for your research...at a certain frequency, and this frequency can indicate behavioral state, for example, if someone is awake...) are well understood, the direct effect of DA binding to a target neuron is more mysterious. Thus, scientists... ms on and 100 ms off), enabling detection of individual peaks of DA release.  Using dLight1 sensors in...during learning and reward consumption in live mice Find dLight sensor here! The GRABDA dopamine sensor Yulong... such that the GFP fluoresces upon DA-receptor binding (Sun et al., 2018). These sensors also exhibit ... mice over the course of Pavlovian conditioning Find the GRABDA sensor here! Overall these sensors are...

  19. Plasmids 101: Antibiotic Resistance Genes

    Type
    Blog Post
    Published
    Jan. 30, 2014, 3:29 p.m.
    ...plasmid, scientists would spend hours to days trying find their correct clones. Additionally, the presence...vice versa). Looking around the lab, you'll likely find many of the antibiotics listed in the table below...Working Concentration** Kanamycin aminoglycoside Binds 30S ribosomal subunit; causes mis-translation Bactericidal...Bactericidal 50-100 ug/mL Spectinomycin aminoglycoside Binds 30S ribosomal subunit; interrupts protein synthesis...Bactericidal 100 ug/mL Bleomycin glycopeptide Induces DNA breaks Bactericidal 5-100 ug/mL Erythromycin...Bactericidal 10-100 ug/mL Tetracycline tetracyclin Binds 30S ribosomal subunit; inhibits protein synthesis... Bacteriostatic 10 ug/mL Chloramphenicol   Binds 50S ribosomal subunit; inhibits peptidyl translocation...

  20. Beyond the Blood Brain Barrier: PHP.B Enters the Central Nervous System Via the LY6A Receptor

    Type
    Blog Post
    Published
    July 23, 2019, 12:46 p.m.
    ...on PHP.B binding in vitro. They found that when LY6A contained C57BL/6J SNPs, PHP.B could bind it, but ...an ELISA to measure binding of PHP.B to a soluble version of LY6A. They saw binding between PHP.B and the...Wilson lab at the University of Pennsylvania independently searched for this unique sequence of DNA and...susceptibility. The Deverman and Wilson Labs independently identified LY6A as a receptor for PHP.B by looking...search on SNPs and small insertions and deletions (indels) most likely to have large phenotypic effects such...but when LY6A contained BALB/cJ SNPs, binding was reduced. The Deverman group then looked to see if PHP....mutation in PHP.B’s unique capsid loop disrupted binding. Collectively these results suggest that PHP.B ...

  21. New Tools Enable CRISPRa for Neuroscience Applications

    Type
    Blog Post
    Published
    July 2, 2019, 12:51 p.m.
    ...our comprehensive CRISPR guide Find CRISPR plasmids and resources Find CRISPRa plasmids ...engineered combinations of transcriptional activators to induce gene transcription at genomic sites targeted by...cDNA libraries. However, using CRISPRa to target individual genes can be relatively laborious because multiple... control over its expression. Constitutive or inducible Cre recombinases can be introduced by AAV or by...in defined substructures of the CNS as well as individual neuronal populations.  The Yang Lab’s AAV plasmids... promoter, which was superior in its ability to induce CRISPRa in primary rat neurons over other promoters...straightforward than generating cDNA for each gene isoform individually. To highlight this advantage, they selected ...

  22. AAV Titers: Where Do They Come From And What Do They Mean?

    Type
    Blog Post
    Published
    Nov. 15, 2017, 3 p.m.
    ...are addressed with ddPCR technology (Hindson et al., 2011, Hindson et al., 2013), which has been shown ...2011): 8604-8610.4. Hindson CM, Chevillet JR, Briggs HA, Gallichotte EN, Ruf IK, Hindson BJ, Vessella RL,...Resources on Addgene.org Find All Available AAV Learn More about Virus Production Find Viral Vector Protocols...value, physical titers don’t necessarily give any indication of the infectious titer. The infectious titer...Although you can optimize your primers for each individual sample, this will reduce convenience and comparability... the assay. This may be due to competition for binding sites between the primers and the AAV genome repeats...Finally, because physical titer does not give an indication of infectious titer, consider titrating each ...

  23. Transferable Skills Guide: Career Planning Resources

    Type
    Blog Post
    Published
    Nov. 14, 2017, 2 p.m.
    ...employer in mind, you may consider starting with indeed.com or monster.com, where you’ll find a variety ...employment within industry or academia; although the ACS career fair is more industry-focused. For another...another industry-geared option, try the Biospace career events page. Biospace holds career events all over...institution’s career page, where you’re likely to find current job openings. A good approach is to do this...of academia, you may need to look a bit harder to find your mentor. This may be a connection you’ve made...and other popular job site webpages. Importantly, find a good mentor that can share relevant experience...www.gradschools.com/get-informed/grad-school-roadmap Find Job Opportunities for Scientists on docjobs.com ...

  24. CRISPR Activation: A Practical Guide

    Type
    Blog Post
    Published
    Aug. 18, 2016, 2:30 p.m.
    ...example, CRISPRa can be used to find potential cancer drug targets by finding genes that cause cell death ...Used with AAV Resources on Addgene.org Find Cas9 Activators Find Cas9 Repressors Catch Up on Your CRISPR...CRISPR activation has been used to differentiate induced pluripotent stem cells (Chavez et al., 2015) into...Additionally, CRISPR-based activation can be made inducible upon environmental triggers. This is done by creating...activators, similar tools were made using other DNA binding proteins such as zinc fingers (ZF) and TAL effectors...by putting the promoter of the gene into a gRNA finder such as WU-CRISPR (Wong et al., 2015)) or our lab....2018.04.011 Polstein LR, Gersbach CA (2015) A light-inducible CRISPR-Cas9 system for control of endogenous gene...

  25. Single-cell tracking of lineage and identity with CellTag

    Type
    Blog Post
    Published
    May 9, 2019, 1:15 p.m.
    ...University in St. Louis provides a combinatorial indexing strategy that simultaneously tracks clonal history...variable region acts as a unique identifier for individual cells. Each cell is labeled with 3-4 tags to ...decreased over time but they were not silenced.    Find CellTag libraries here!  Applications of CellTag...reprogramming of mouse embryonic fibroblasts (MEFs) into induced endoderm progenitors (iEPs), a self-renewing cell...takeaways of CellTag CellTag is a combinatory cell-indexing tool that combines scRNA-seq and lineage tracking...identity changes, such as the differentiation of induced pluripotent stem cells or the clonal evolution ...cancer. If you’re looking to use CellTag, you can find the CellTag libraries at Addgene. The Morris Lab's...

  26. 10 Great Guest Posts We're Thankful For

    Type
    Blog Post
    Published
    Nov. 18, 2016, 3 p.m.
    ...post are engineered to bind RNA sequences derived from Zika virus and, upon binding, produce reporter proteins...posts written by Addgene depositors. Below you’ll find brief summaries of several excellent guest posts... post, Luke Lavis from Janelia Research Campus reminds us that, although fluorescent proteins are incredibly...used in a wide variety of experiments. Read more to find chemical fluorophores for your next experiment. ...virus biology while leaving virulent components behind. Minigenomes are stripped down viral genomes containing...post for potential applications of R-bodies and to find a video of them in action. AAV: A Versatile Viral...Medical School describes how AAVs function and indicates important things to consider if you’re getting...

  27. Fluorescent Proteins 101: Photoactivatable Fluorescent Proteins

    Type
    Blog Post
    Published
    April 25, 2017, 2:30 p.m.
    ...Addgene.org Find Photoactivatable FPs in Empty Backbones in the Michael Davidson Collection Find Photoactivatable...from a dark state to a bright fluorescent state (“kindling”), and from a bright state to a dark non-fluorescent... In 2008, scientists used mutagenesis screens to find a version of Dronpa that doesn't become inactivated...photoactivation. A high contrast ratio usually indicates that a PA-FP has a low level of spontaneous fluorescence...photobleaching and phototoxicity that standard FPs might induce though their continuous excitation. By generating...photoswitching between two fluorescence wavelengths. Indeed, within the world of super resolution microscopy...localization information in cells. A point to keep in mind- although PA-FPs are vastly improved over the conventional...

  28. How to Deposit Your Plasmids with Addgene

    Type
    Blog Post
    Published
    Feb. 27, 2024, 2:15 p.m.
    ... 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 ...information will make it easier for scientists to find your plasmid by type. Figure 8: Add vector backbone...

  29. From Lab Bench to Leadership: Transitioning into Management as a PhD

    Type
    Blog Post
    Published
    Jan. 21, 2025, 2:15 p.m.
    ... PhDs Making the Leap to Industry." He successfully transitioned to industry after his PhD and a couple... PhDs face when transitioning from academia to industry is whether to stay hands-on in the lab or pivot...exploring alternative career trajectories within the industry, but the transition wasn’t easy - I had to reinvent...assistant. However, I recognized the need to shift my mindset to empower my team and allow myself to focus on... rather than the day-to-day details. Ignoring individual differencesManaging a team isn’t “one size fits...a diverse team where some members thrived on independence, while others required more guidance and structure...learned that my approach needed to adapt based on individual needs. For instance, I had one colleague who ...

  30. GPCRs: How Do They Work and How Do We Study Them?

    Type
    Blog Post
    Published
    Feb. 11, 2021, 2:15 p.m.
    ...works GPCR signaling is initiated when a ligand binds to the extracellular surface of the GPCR. This results...from the Gβγ dimer. The Gα and Gβγ subunits then induce or inhibit intracellular signaling cascades as .... Ligand dissociation from the GPCR allows for binding of a new inactive heterotrimeric G protein complex...identify some of these unknown ligands and their GPCR binding partner, Bryan Roth’s lab created an open source...activation for more than 300 GPCRs. In this assay, binding of an agonist recruits a beta-arrestin-TEV protease... discover new ligands for unknown GPCRs. You can find the entire PRESTO-TANGO kit at Addgene! TRUPATH:...were first mutated via site-directed mutagenesis to bind non natural ligands. These non natural ligands however...

  31. The Many Reasons for Irreproducible Research- and a Vaccine to Eradicate It

    Type
    Blog Post
    Published
    Jan. 30, 2020, 2:15 p.m.
    ...question after credible attempts to replicate major findings could not be replicated by other researchers.There...replicate.  Why is replicating previous research findings so difficult to do?  There are four reasons that... to present only the most novel or significant findings, and combined with powerful cognitive biases such... such as motivated reasoning and hindsight bias, we may fool ourselves and our readers into believing ...research practices (QRPs) can quickly lead us to find statistically significant results that are not robust... not come out the same as an original research finding we can be more certain that some important, previously...makes a big difference for the robustness of the findings.   How do we make research more reproducible?...

  32. Back to Bacteria: CRISPR gRNA Multiplexing Using tRNAs

    Type
    Blog Post
    Published
    June 2, 2015, 6:06 p.m.
    ...100% indel frequency at both targets (35% and 76% biallelic), compared with only 44% and 66% indel frequency...recognition and cleavage. This sequence flexibility indicates that gRNAs could be incorporated into these constructs...the 5’ end. PTG construct schematics. Scissors indicate cleavage sites. After confirming gRNA cleavage...cleavage, they examined gRNA expression levels and indel generation for two PTG constructs in rice protoplasts... allowing them to examine the frequency of both indels at each locus, as well as large deletions. They...deletion with 4-20% frequency at the four loci, indicating that Cas9 can be guided to at least 8 distinct....1073/pnas.1420294112. Epub 2015 Mar 2. PubMed. Find the plasmids from this publication at Addgene ...

  33. dTAG - You're it!

    Type
    Blog Post
    Published
    June 21, 2018, 2:06 p.m.
    ...small molecules that bind these target proteins to other small molecules that bind an E3 ligase. These ...molecules by conjugating a CRBN-binding ligand (thalidomide) to an FKBP12F36V binding ligand (ortho-AP1867) via...E3 ligases such as cereblon (CRBN) or von Hippel-Lindau (VHL) to bring the endogenous degradation machinery...selectivity, overcoming resistance to inhibitors, and inducing prolonged biological effects. While targeted ...oncoproteins that lack selective small molecule binders. To overcome this limitation, we created a generalizable... FKBP12F36V-tagged proteins. The dTAG molecule induces ternary complex formation between the FKBP12F36V-fusion...and the ubiquitin proteasome machinery through binding with cereblon, leading to rapid target degradation...

  34. Antibodies 101: Selecting the Right Antibody

    Type
    Blog Post
    Published
    March 15, 2022, 1:15 p.m.
    ...those of antibody-independent assays such as mRNA data from transcriptomics; independent antibody strategies... internet search for the required antibodies and find….hundreds of options. With limited time and budget...immunoprecipitation, the epitope may not be accessible for binding and the antibody will not work. Whenever possible...more detailed data on the assay methods. You might find that while the website highlights a variation of...fluorophore, or a conjugated secondary antibody that will bind to your primary antibody. If you are trying to visualize...applications use either direct (primary-conjugated) or indirect (secondary) methods. In those cases, remember ...the antibody cross-reacts with. Ideally you will find an antibody that has been validated in the species...

  35. To Each HIS Own

    Type
    Blog Post
    Published
    March 2, 2023, 2:15 p.m.
    ...interest flowed through. The protein of interest binds to the metal ions, becoming immobilized itself. ...containing resin. The protein of interest (purple) binds to the Ni2+ and is immobilized along with a polyhistidine...purification, however, is the propensity for nonspecific binding of untagged proteins. Despite being a relatively...histidines are present in most production systems and can bind and co-elute with the protein of interest (Bornhorst...be high enough to disrupt contaminant-metal ion binding without eluting the target protein and needs to...substrates along with stringent wash conditions.   Find polyhistidine tag plasmids here! Where should I ...Unlike other affinity purification methods where binding relies on a specific protein conformation, the ...

  36. Hot Plasmids: Spring 2025

    Type
    Blog Post
    Published
    June 3, 2025, 1:15 p.m.
    ... articles. Here's what you'll find in this post: Glutamate indicators with increased sensitivity and...viral preps targeting interneurons Glutamate indicators with increased sensitivity and tailored deactivation... for a variety of synaptic imaging applications. Find iGluSnFR4 plasmids here! Aggarwal, A., Negrean,..., Iyer, R., … Podgorski, K. (2025). Glutamate indicators with increased sensitivity and tailored deactivation...and AAV plasmid sets launching later this year!  Find Addgene's own Lentiviral backbone plasmids here!... 5% and 11%. It has higher efficiency and lower indel formation than methods relying on homology-directed...scarless, unlike previous integrase-based approaches. Find STITCHR plasmids here! Fell, C. W., Villiger, L...

  37. Is this the right place for me? 8 tactics for choosing a lab

    Type
    Blog Post
    Published
    Oct. 2, 2018, 12:56 p.m.
    ...options critically. How will it feel when you find it? If you find the right lab, some days it won’t even feel...scientists seem to toss data-driven reasoning out the window when making decisions. We think scientists make...success. Gather data on the lab choices How do you find out what the lab is really like? Gather data of ...boss You sign on to a lab for the mentoring only to find out that the lab head is an absentee advisor. They...character traits and skills that you hope to grow.  Find mentors who take their responsibility to train you...meaningless tasks, removal of responsibility, repeated reminders of blunders, setting target up to fail, shifting...(topic and/or technical aspects) applicable to industry if this path interests you  (e.g. human disease...

  38. Hot Plasmids - October 2020

    Type
    Blog Post
    Published
    Oct. 13, 2020, 1:15 p.m.
    ...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: Tools for labeling excitatory inhibitory...respectively. Once expressed in the neuron, the FingRs bind to the endogenous synaptic proteins allowing them...inhibitory or excitatory synapses or that are Cre-inducible, researchers can explore synaptic integration ... show a high proportion of blue fluorescence, indicating cell proliferation. BaF3 cells maintained in ...in high IL-3 showed mostly blue fluorescence, indicating a quick cell cycle. Cells that had IL-3 withdrawn...a few highlights from recent CRISPR plasmids. To find all of the CRISPR plasmids available from Addgene...

  39. Split-BioID: An Improved Method for Studying Protein-Protein Interactions

    Type
    Blog Post
    Published
    May 1, 2018, 1:57 p.m.
    ...at Addgene.org Find plasmids from the Bethune Lab Find Plasmids from the Roux Lab Find Plasmids for FRET... particular protein  complex as opposed to an individual protein. The Bethune Lab has overcome this limitation...directly interact with the bait from those that indirectly interact or from proteins that are just in the...labeling. BioID requires 6-24 hours for tetracycline induced expression of the BirA* fusion protein and saturation...Schopp et al allowed 24 hours for tetracycline induced expression and biotinylation of interacting proteins... PMID: 28585547. PubMed Central PMCID: 5467174. Find the Split-BioID Plasmids Here! Munter, S.D., Görnemann...fragmented at a different site than in Schopp et al. Find these Split-BioID Plasmids Here!   Burke, B., Kim...

  40. What's New in CRISPR - May 2019

    Type
    Blog Post
    Published
    May 28, 2019, 12:49 p.m.
    ...Addgene.org Read our CRISPR guide Find CRISPR plasmids by function Find recently deposited plasmids ... but we hope that this blog series will help you find new CRISPR tools for your research! This time: ...control. Read the paper in Nature Communications Find the E. coli genome-wide CRISPR inhibition library...fold. Read the paper in Communications Biology Find the Cas9-PCV and PCV-Cas9 plasmids Selective curbing... editing efficiency. Read the paper in Nature Find the SECURE base editors All-in-one AAV delivery... in zygotes. Read the paper in Molecular Cell Find the Nme2Cas9 All-in-One AAV constructs CRISPR interference...essential C. albicans gene. Read the paper in mSphere Find the dCas9 and dCas9-Mxi1 plasmids If you have a...
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