...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...

...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...

...at the 5′ or 3′ end of the sgRNA (i.e. TOP1 or TOP2 constructs; see Supplementary Note 3 in the article... many potential applications. Polstein LR & Gersbach CA, Nat Chem Biol 2015 Mar;11(3):198-200. SunTag...presented Archaerhodopsin 3 (Arch) as a new rhodopsin based voltage indicator for use in the detection ...luciferase mRNA (3’UTR AUUUA [5 consecutive]) and protein (C-terminal CL1 & PEST). These modifications ensure that...protein (NLS-PCP-GFP) a RFP protein that binds to the 3’ UTR of the reporter mRNA via an MS2 coat protein ..., (2) tamoxifen-inducible CreERT2 gene deletion, (3) simultaneous expression of cDNAs and shRNAs with ...8 constitutive promoters, 2 includible promoters, 3 polyA terminators and various pieces for inducible...

...bind to the anti-Protein B antibody (Fig. 3)    Figure 3: Recommended secondary antibodies for rat...published, knockout validated, and work in your application. But…they are the same isotype and therefore ...are identical for antibodies within certain classification groups called isotypes.  Why does isotype matter...

...spring of 2015 and since that time have received about 3 project proposals per month.  We are currently focused...greater chances for success.  Build a team of 2 or 3 Don’t go it alone.  You’ll find that no matter how...launching a company without partners.  You need a dedicated team that you know well and trust.  This will ...Chowdhury and Clinton Buell, who have very diverse educational backgrounds (an essential component to any successful...

...to supply it with another plasmid.   Figure 3: Comparison of Multiplex Strategies including Standard...For example, if you were to develop an array using 3 distinct spacer-repeats, you could easily create 7...a subsequent curing protocol that requires only 2-3 hours incubation. Kondo Lab multiplexed base editing...PMC4231726. Find plasmids from this paper at Addgene. 3. Sakuma, Tetsushi, et al. “Multiplex genome engineering.... Using multiplexed gRNAs can target multiple locations in the genome to modify, whether it be editing... use is pX333 from the Ventura lab. pX333, a modification of pX330, contains humanized wtCas9 and two ...thus marking cells that have undergone genomic modification. In this system, Cas9 must be supplied on a ...

...decided to pack it in. I had just started month 8 of my 3 year Post-Doctoral contract at the University of Cambridge...this job specifically looked for a researcher with 3 years+ of cancer research experience, a PhD or a Master...there a move with either promotions or further education may allow you to transition into various realms...

...tumor-infiltrating lymphocytes of that tumor. In Figure 3 you can see how I gated out my lymphocyte population...look for them on the FSC-A/SSC-A plot. Figure 3: (A) With the acquisition voltage (not shown) used...gates while some like to make looser ones (Figure 3). A more “liberal” gate in the beginning should not... you with two measurements: forward scatter (FSC) and side scatter (SSC). To acquire these measurements...exclude all duplicate events (two cells stuck together) from your single cells, as duplicates can’t be reliably...types of scattering that occur when a cell is pulsed by the laser. Within each type of scattering, you also...to keep it short though, promise! FSC and SSC: scattering To digitally extract your cells based on their...

...exchange, the government protects (and often funds [3]) scientists as well as other citizens. Thus civilization...depending on the number of people supporting it.  3) Linking scientists to one particular political party...request of this administration promises more cuts (3, 12, 13, 27). In order to achieve the breakthroughs...sister locations on April 22, 2017, scientists and non-scientists alike will march to advocate for science...already come to pass. It may only be by advocating for science education and literacy that this changes.  2...marching for. I will be marching to advocate for science education and literacy as I believe people that...communities, microbial interactions, and science education.  Disclaimer: The views presented in this article...

...Chapter 3: Finding the Right Antibody for Your Experiment.” Whatever your antibody application question...resource for anyone looking to use antibody-based applications in their research.  Like all our eBooks, the...through the process. If you’re confident in your application and protocols, but can’t quite seem to find an...

...customized sequence specificities." Nature communications 3 (2012): 1147. Cooke, Amy, et al. "Targeted...the core of a Shine-Delgarno sequence found at the 3’ end of the leader sequence. Using a variety of GFP...create custom RNA binding proteins in as little as 3 days. Abil et al., 2014 show that you can fuse custom...well as protease sites to facilitate protein purification. We hope this kit will allow you to quickly ...for a variety of synthetic biology or protein purification studies in E. coli. 800 and 1900 Series If you...polycistronic E. coli expression constructs. Some applications of plasmids from this kit include the production...and Charles E. Melançon III. "Detection and Quantification of Ribosome Inhibition by Aminoglycoside Antibiotics...

...abundant incorporation of cysteine in their proteomes (3). This led to the eventual discovery of a naturally...which can be specifically recognized by the Sep-tRNA. 3. An expression plasmid encoding the ORF to be expressed...PMID: 25514926. PubMed Central PMCID: PMC4383998. 3. Li, Tong, et al. "Cysteinyl‐tRNA formation: the last...most inclusive databases of post-translational modifications, identifies a staggering ~250,000 phosphorylation...steric and charge-based nature of the phosphoryl-modification; they are both much smaller, and carry only ...thus cannot reflect the true state of protein modification within the context of its appropriate regulation...codons (TAG), replacing them with TAA (7). This modification allowed the deletion of release factor 1, the...

...Figure 3 shows a histogram for a yes-no dot plot, like the one shown in Figure 1.  Figure 3: An example...to the right indicates a higher signal, or upregulation, and a peak to the left indicates a lower signal...anything to the right of it, marking it as GFP+. Quantification for such graphs could simply be the percentage...case for you, switching to a contour plot should indicate where the two populations diverge. The smear ...place your gate based on the dot plot would be complicated.  Figure 2: (A) a smeary dot plot showing...Figure 4 compared to the dot plot in Figure 2. Quantification using MFI makes it easier to identify and study...addgene.org Addgene's Antibody Guide rAb Affinity Purification Protocol Video rAbs in the Addgene repository...

...used saving the researcher both time and resources (3). Still, researchers generating transgenic animals..., called dideoxynucleotides (ddNTPs), lacking the 3’ hydroxyl group required for the extension of a DNA...Medicine 307.1 (1982): 30-32. PubMed PMID: 6176866. 3. Hoebeeck, Jasmien, Frank Speleman, and Jo Vandesompele...comparison to a control sample. Southern blotting applications range from confirming the results of cloning...interest by recording DNA abundance throughout the amplification process, rather than just at the end as in standard...experiments allowed researchers to identify the location of genes. With this knowledge, researchers were...was added and when. Compiling the identity and location of a given nucleotide, a researcher (or a simple...