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...sequence for recognition by QCascade; although 5’-CC-3’ is one of the best PAMs for VchINT, many other PAMs..., by expressing multiple crRNAs in parallel (Fig. 3), multiplexed insertions into multiple distinct targets... or isolated (Rubin et al., 2020).  Figure 3: Multiplexed insertions with Type I-F INTEGRATE using... and have been adapted for a wide variety of applications - as mutagenesis tools for genetic studies (... The process also creates a 5-bp target-site duplication (TSD), a hallmark characteristic of the Tn7-family...the new streamlined VchINT system was shown to catalyze insertion of mini-transposons up to 10 kb in size...at one or multiple desired genomic loci. Many applications of these systems have already been demonstrated...

... different systems could be combined effectively (3). In Figure 1 for instance, the DBD of the fusion ...activator, bacterial LexA DBD and the lacZ reporter gene (3, 4), there are now many variations, including many...Science. 1986; 231:699-704. PubMed PMID: 3080805. 3. Brent, R., and Ptashne, M. A eukaryotic transcriptional... can often be accounted for using appropriate replicates and controls (see Tips below), and by validating...two-hybrid screening Minimizing false positives: Run replicates of the experiment to reduce the likelihood of...expression and correct folding, post-translational modification, protein degradation, access to the nucleus ...co-expression of the enzyme responsible for the modification in the assay host strain can help. In order...

...program to design Gateway plasmids.    Figure 3: Method A to create an entry clone: recombination ...Gateway cloning efficiency can reach >99%.   STEP 3: Express your Gene of Interest! Be sure to verify ...2003 Feb;Chapter 5:Unit 5.17. PubMed PMID:18429245. 3. Ptashne, M. (1992). A Genetic Switch: Phage (Lambda...attP sites of the donor vector. This reaction is catalyzed by the BP Clonase enzyme mix and generates the...sites of the destination vector. This reaction is catalyzed by the LR Clonase enzyme mix. As a result, an ...carry different antibiotic resistance markers (indicated here by plasmid color), allowing you to easily...is simple -  no restriction, ligation or gel purification steps are required! Multiple fragment cloning...

...genome which was composed of the wild-type 5’ and 3’ LTRs, the ψ sequence, a part of the env gene containing...exception of the env gene (packaging plasmid), and 3) the vesicular stomatitis virus G protein (VSV-G) ... been described (protocol 1, protocol 2, protocol 3) to find the one that will give you the best production...Science 272.5259 (1996): 263. PubMed PMID: 8602510. 3. Yasutsugu Suzuki and Youichi Suzuki (2011). Gene ...mammalian cells often relied on chemical (CaCl2, cationic liposome) or electrical (electric current forming...particles and the virus particles produced could not replicate. However, Mother Nature found a way to re-create...packaging plasmid forming a vector that could create replication competent virus. Even though this system was ...

... microscopy. Nanobodies can be labeled with up to 3 dyes, yielding similar signal strength to a standard...nanobodies are useful for colocalization labeling of up to 3 targets (Pleiner et al figure 4D). Unlike large secondary...PMID: 26633879 PubMed Central PMCID: PMC4755751   3. Muyldermans, Serge. “Nanobodies: natural single-domain...in bacteria and extracted with common protein purification methods. Read on to learn more about nanobodies..., Alexa Fluor 568, or Alexa Fluor 647 before application to the sample. Staining with this multi-color...PubMed PMID: 29263082 Find plasmids from this publication at Addgene. 2. Bates, M., Böhning, M.I., Chug...

...labels than fusions to 3 or more HA tags. 4. High-resolutions microscopy As indicated above, smFPs can be...moleculare were better resolved with smFP_FLAG than GFP.   3. Enhanced ability to distinguish weakly expressed ...brain". Oxford University Press, New York. pp 719. 3.Rizzo, Mark A., Michael W. Davidson, and David W. ...controlled by collections of neurons that are intricately wired into circuits through synaptic connections...photostable than GFP. In addition, antibody-based amplification of their signals is weak and increases background...

...possible because there are 4 bases (A, T, C, and G) and 3 positions in each codon. There are therefore 64 possible...possible codons but only 20 amino acids and 3 stop codons to encode leaving 41 codons unaccounted for....PMID: 15678157. PubMed Central PMCID: PMC1299251. 3. Emilsson, Valur, and Charles G. Kurland. "Growth ...ribosome may therefore stall at inappropriate locations or fail to make it through the entire transcript...generally segregate with the cell pellet during purification procedures. Even if you produce a large amount...

...vector is located in the 3’ end of the multi-cloning site, and the reverse primer is located in the 5’...correct size (vector + insert). You should screen 3-4 colonies to ensure that you find the correct product...Restriction Enzymes + - PCR Amplification + + Digestion + + Gel Purification + - Ligation + - Transformation...Molecular cloning is the isolation, insertion and amplification of a recombinant DNA without sequence alteration... multi-step process consisting of digestion, purification, ligation, and transformation. While restriction...sequences for base pair overhangs, insert and vector purification, and, most importantly, excessive time requirements...is ligation independent, it does not require purification of insert or vector products, nor does it require...

...Na+ and other cations. B) Cortical slice of HcKCR1-EYFP and tdTomato expressed layer 2/3 neurons in mouse...know. We’d love to hear from you!   Fig. 3: Image of Addgene’s western blot for the myc-tagged ...what you'll find in this post: Improved voltage indicator: JEDI-2P Novel class of light-gated potassium...-Myc [9E10] now available! Improved voltage indicator: JEDI-2P  by: Brian O'Neill  The François St-Pierre...have successfully developed an improved voltage indicator (GEVI) called JEDI-2P. Using a custom, high-throughput...predecessor GEVIs (called ASAP 1 and 2), they found an indicator that is optimized for 2-photon imaging of voltages...screen for improved genetically-encoded voltage indicators (GEVIs) based on the ASAP sensors. The resultant...

...protein. Nature Communications, 14(1), Article 1. https://doi.org/10.1038/s41467-023-39655-3  Wilhelm, J....depending on the needs of your experiment (Figure 3). And using self-labeling tags makes updating your...ligands at different time points.   Figure 3: Single-molecule tracking of actin filament assembly-regulating...Imaging and Protein Analysis. ACS Chemical Biology, 3(6), 373–382. https://doi.org/10.1021/cb800025k  Miller...Few Complicated Non-natural amino acids 1 Various Many Complicated   Applications Organic...new green fluorescent protein, some affinity purification tags, another version with that same red fluorescent... an enzyme that has been mutated to block its catalytic activity and instead become trapped in a covalent...

... DNA by joining 5 ́-phosphorylated DNA termini to 3 ́-OH DNA termini (1-4). In vitro, ligases (notably...the ligation junction (the base pair providing the 3 ́-OH terminus to the ligation) than on the downstream...reviews 106.2 (2006): 687-699. PubMed PMID: 16464020.  3. Ellenberger, Tom, and Alan E. Tomkinson. "Eukaryotic...Colette, Veronique Bailly, and Walter G. Verly. "Nicks 3′ or 5′ to AP sites or to mispaired bases, and one-...or DNA base pair mismatches (8-11). For some applications, there cannot be any end-joining activity, and...regions. Why use a high fidelity ligase? Numerous applications have been developed to take advantage of the...Taq and other thermostable DNA ligases. These applications detect specific nucleotide sequences with high...

...Escherichia coli and Agrobacterium tumefaciens. 3 Biotech, 13(3), 91. https://doi.org/10.1007/s13205-023-03507...plasmid RK2. Molecular & General Genetics: MGG, 152(3), 129–135. https://doi.org/10.1007/BF00268809  Prior...ori2, ori3) and replication initiation proteins (RepA, RepB) necessary for replication in various hosts...the ori and allow for replication. However, if plasmids have their own replication machinery such as Rep...different hosts that a plasmid can transfer to, replicate in, and persist in. Some plasmids have a narrow... while others have a broad host range and can replicate and stably maintain the genes they carry among.... These features are: The plasmid origin of replication (ori) contains structural elements that are versatile...

...clearly described in a journal publication. Plasmids will be released 3 years after the deposit date. ...other scientists to use? We take this Hold for Publication policy seriously and appreciate the trust you...of July 1, 2025, we are updating our Hold for Publication policy with two changes that will help us ensure...keep things moving internally. And remember, a publication isn’t required to share your plasmids! You can...can make your deposit available before publication and we can update the plasmids’ citation when the paper...

...it into four, using the location of the cell populations as a guide  (Figure 3.) You can see that in our... but no cells that are A+B-.      Figure 3: It's often easier to identify different populations...series of one- or two-marker plots, like in Figure 3 above, until the population of interest has been successfully...done using side scatter (SSC) and forward scatter (FSC), which measure light that “scatters” past the cell...be used for this purpose.  Pro tip! The forward scatter is a proxy for diameter and volume of the cell,...the exclusion of doublets and debris. The side scatter roughly measures granularity, a proxy for nucleic... will have numbers by gated populations; this indicates the percentage of total events contained in the...

...nickases; D10A creates 5’ overhangs and H840A creates 3’ overhangs in a PAM-out design.  Exploring nickases...Addgene Blog Learn about Homology Directed Repair 3 Tips to Improve HDR Efficiency How to Design Your ... a Cas9 nickase and two gRNAs.  For nickase applications, a common question is: how should the gRNAs ...

...improve AAV yields. Although we only investigated 3 serotypes it’s possible that this approach will be...bioengineering 42.5 (1993): 601-610. PubMed PMID: 18613082. 3. Oyaas, K., et al. "Hybridoma growth and antibody ...AAV yields. Given the wide range of downstream applications scientists rely on our AAV preparations for,...

...evidence you need. For example, as shown in this study [3], hippocampal neurons are activated when the mouse...PMID: 25242321. Pubmed Central PMCID: PMC4181379. 3. Kheirbek, Mazen A., et al. "Differential control ...retrieval strategies for remote memories. Cell. 2011;147(3):678-89. Pubmed PMID: 22179551. Pubmed Central PMCID...EthoVision. Consult the literature to find all the applications of optogenetics relevant to your field. Ultimately...Inhibition of neuronal activation is a little more complicated since basal neuronal activity in your brain region...technology is improving and may one day reach the sophistication of mouse transgenics. Everything that has been... and memory in opioid addiction and science communication/science writing. Derek is also a pianist and...

...injection step! The entire protocol takes about 2-3 weeks but requires less than eight hours worth of ...Heppert JK, Higgins CD, Goldstein B. Genetics. 2015 Jun 3. pii: genetics.115.178335. [Epub ahead of print] PubMed...genomic modifications, is very robust in C. elegans, and the methods for HDR-mediated modification continue...After self-excision, the resulting loxP scar is located in this synthetic intron, and only the fluorescent...use and highly efficient for C. elegans genome modification. Figure 2: Schematic from Dickinson et...permission from the Genetics Society of America. Applications of this system Dickinson et al. have developed...system could be used to make many other genome modifications, including targeted mutations. SEC-based systems...

...delivery." Elife 3 (2015): e04766. PubMed PMID: 25497837. PubMed Central PMCID: PMC4383097. 3. Kim, Sojung... reagent. This interplay falls into two broad categories and thus, causes two types of editing outcomes...altering its function). HDR is crucial for certain applications, for example, correcting the allele that causes... dissociation rates and discovered that the catalytically active form of the nuclease dissociates very...we measured identical dissociation rates for catalytically inactive dCas9 molecules. We followed these ...Our in vitro results also demonstrated that catalytically inactive dCas9 produced a “bubble” structure...nucleofection The in vivo editing experiments in this publication were performed using a technique called nucleofection...

...but to also destroy the phage upon re-exposure (2, 3). More specifically, specialized Cas proteins snip...preventing the Cas nucleases from re-identifying it (3). Surprisingly, few genes have been identified that...distinct types and subtypes using polythetic classification based on evolutionary similarity (5). Type ...2013 (7, 8). Nine months later, more than 1500 publications describe work to improve the tool’s specificity...various species and describe an assortment of applications. Despite the ubiquity of CRISPR/Cas toolkit,... multiple plasmids used for various platform applications. For more information on CRISPR/Cas, check out...for your research. Do you have a cool idea or application that involves CRISPR/Cas technology? Let us know...