...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 ...resources on Addgene.org Learn about Addgene's viral service Learn about viral production Find viral protocols... By now you have probably heard about Addgene’s viral service but did you know that Addgene also performs... approach can be adapted by vector cores and laboratories alike as an easy and inexpensive method to improve...Effect of medium osmolarity on hybridoma growth, metabolism, and antibody production." Biotechnology and ...

...portion of our careers. In that time we may have only 2-3 formal supervisors that will provide guidance and ...Connie Cepko to learn about her mentoring style. Top 7 questions scientists ask me about mentoring In this... answers to the top questions scientists ask me about mentoring. Here they are: What makes a good Mentor... Where can I find a Mentor? What is this I hear about “group” or “peer” mentoring? How do I ask someone...a good science mentor? I started a conversation about many of these topics with the researchers who joined...often one that devotes time and energy to thinking about how to be a great Mentor.    Check Out Angela Depace's...questions in future posts. If you have a question about mentoring that you want us to cover, let us know...

...Resources on the Addgene Blog Learn about Homology Directed Repair 3 Tips to Improve HDR Efficiency How...nickases; D10A creates 5’ overhangs and H840A creates 3’ overhangs in a PAM-out design.  Exploring nickases...

...slice of HcKCR1-EYFP and tdTomato expressed layer 2/3 neurons in mouse. C) Action potential frequencies ...us know. We’d love to hear from you!   Fig. 3: Image of Addgene’s western blot for the myc-tagged...Brian O'Neill  The François St-Pierre lab and collaborators have successfully developed an improved voltage...

...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...Article 1. https://doi.org/10.1038/s41467-023-39655-3 Wilhelm, J., Kühn, S., Tarnawski, M., Gotthard, G....but it’s real! In this post, we’re going to talk about the power and flexibility of self-labeling protein...ready-to-use without any cloning at all! As mentioned above, several variants of these tags have been developed...Illuminating Choices blog post for more discussion about choosing a dye or ligand to use for your experiment...

...series of one- or two-marker plots, like in Figure 3 above, until the population of interest has been successfully...location of the cell populations as a guide  (Figure 3.) You can see that in our dot plot, we have A-B- cells... but no cells that are A+B-.      Figure 3: It's often easier to identify different populations...  Of course, there are many more things to learn about when diving into flow cytometry data. There’s different... ways to use FSC and SSC, as briefly alluded to above. There’s many variations in how one can gate, select...

...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...of the important considerations you should think about when developing optogenetics behavioral experiments...scientific research is that the field benefits from collaboration and the exchange of ideas. The rapid spread ...versatility and standardization of genetic tools in the laboratory mouse, most optogenetics is performed in this...

...bind to the anti-Protein B antibody (Fig. 3)    Figure 3: Recommended secondary antibodies for rat...flexibility needed for multiplexing. In the case above, you can’t costain with your desired antibodies ...multiplexing with chimeric antibodies. In the case above you should use an anti-rat IgG1 secondary antibody...this blog post has answered all of your questions about the utility of isotype conversions and chimeric ..., Bright, S. M., Robinson, M. K., Graham, E. T., Sabo, J. P., Owens, R., King, D. J., Norris, S. H., &...

...one injection step! The entire protocol takes about 2-3 weeks but requires less than eight hours worth...Heppert JK, Higgins CD, Goldstein B. Genetics. 2015 Jun 3. pii: genetics.115.178335. [Epub ahead of print] PubMed... this species, namely, the robust HDR mentioned above, an accessible germline, and multiple selectable...modular system that requires very little hands-on labor and could facilitate the tagging of every protein...

...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...for Dummies”. The fastest way to learn something about marketing would be to start reading a broad simple...introduction to the financial world. It teaches you about selling products which is an important part of the...Seán on twitter @Macfhearraigh.    Read more posts about Scientific Career Development ...

...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...plasmids may have high copy numbers and reduced metabolic load (Meyer 2009). Figure 1: Idealized ...

...at the 5′ or 3′ end of the sgRNA (i.e. TOP1 or TOP2 constructs; see Supplementary Note 3 in the article...Polstein LR & Gersbach CA, Nat Chem Biol 2015 Mar;11(3):198-200. SunTag system for single molecule imaging...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... 2012, Adam Cohen's lab presented Archaerhodopsin 3 (Arch) as a new rhodopsin based voltage indicator ...shorten the half-life of both the luciferase mRNA (3’UTR AUUUA [5 consecutive]) and protein (C-terminal...

...without investing time and labour on reagent preparation in the lab. Figure 3: csRNP-assisted epitope tagging...-in for 96 different genes in a single go (Figure 3). We were able to tag >30 genes/plate in the first...we could tag >60 transcription factors in a short (3-week) time window; this was possible because reagents...PMID: 29638216. PubMed Central PMCID: PMC5947990. 3. Jarvik JW, Telmer CA. 1998. Epitope tagging. Annu...ChIP-seq methods provide a wealth of information about how protein networks work in cells. A major bottleneck...two-part cr/tracrRNAs for CRISPR experiments (in collaboration with Integrated DNA Technologies, USA). These...first crRNA we designed for ~250 genes, we have had about 30% tagging success, although many of these genes...

...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...multiplexing options, but also check out our blog post about multiplexing with Cpf1.   Figure 1: Multiplexing...assembly.  Unlike some of the plasmids described above, this vector does not contain Cas9, so you’ll need...similar to the Csy4 polycistronic system described above, except that they use an endogenous nuclease system...

...spring of 2015 and since that time have received about 3 project proposals per month.  We are currently ...greater chances for success.  Build a team of 2 or 3 Don’t go it alone.  You’ll find that no matter how...

...students who are passionate about communicating science.  ComSciCon's events have 3 main goals: To help graduate...succeeded in communicating the information you intended. 3. Join the scicomm community Like most aspects of science...  If someone asks a question about something they did not understand about your presentation, question ...important and relevant to them.   When you think about it, virtually every milestone in a scientist's career...scientists learn from advisers and other mentors by collaborating as they craft sentences for abstracts and presentation...  If someone suggests a change to a slide, think about whether the same principle applies to other slides...much bigger job than merely telling your audience about the work you've done. If you want to maximize the...

...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... with a testable hypothesis - an educated guess about how some part of the world functions. Experiments...governments exist in reality and science is how we learn about reality. Formally, a government can be defined ... More concretely, scientists gather information about reality informing people and their governments on...people to rally for science, others are concerned about possible unintended consequences of a march. Three...

...some impressive videos of these interactions (Ref 3). You can find useful tips on in vivo imaging in our...Research 9.12 (2016): 895-905. PubMed PMID: 27913448. 3. Cai, En, et al. "Visualizing dynamic microvillar ...Cancer, Inflammation and Immunity and learn more about “... recent findings in basic immunology, cancer-immune...curing cancer in cross-disciplinary projects and collaborations. Summarized by Professor Michael Karin from...This was followed by a talk from Professor Karin about alcohol and obesity as driving factors for chronic...got a chance to talk with Dr. Friedmann-Morvinski about tools used to analyse the tumor microenvironment...community, contact [email protected] to find out more about our free deposit and distribution service! References...

...-lantern emitted yellow-green light and exhibited 3-5 fold increased brightness over previous BRET probes... single-cell and whole-body imaging.” Nat Commun. 3 (2012): 1262. PMID: 23232392 PMCID: PMC3535334 Find...Since their first publication in 2012, the Nagai laboratory has assembled a collection of multicolored nano-lanterns...Nano-lanterns, Takai et al. continuously imaged cells for about 4 hours! The latest enhanced Nano-lanterns (eNL)... Nano-lanterns and your experiments The Nagai laboratory has a done a wonderful job characterizing Nano-lanterns...Additional Resources on the Addgene Blog Learn about Photoactivatable Fluorescent Proteins Check Out ...

...chloro-3-indolyl-β-D-galacto-pyranoside) into galactose and an insoluble blue pigment (4-chloro-3-brom-...antibiotic selection here at Plasmids 101, we can talk about an even more specific method of screening your cloning... plasmid is a great start when cloning, but how about being able to choose those that contain plasmid ... a widely used method to do just that! (To read about other screening strategies, check out our screening... called α-complementation. The system described above was put to practical use in the following way. Scientists...

...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...lymphocytes in a mouse tumor sample You made it! So much about the theoretical background. Let’s dive into the ...often choose a looser gate here. I am not worried about including irrelevant events in my analysis, as these...population, acquired from the previous gating pipeline, about 53% of my cells are dendritic cells and ~13% are...ve covered in this blog post. First, you learned about the types of scattering that occur when a cell is...

...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... one of the MEK1 phosphomimetic residues in the above study with phosphoserine (to create a pSer/phosphomimetic...non-phospho species on PhosTag gels as described above. Reagents and protocols Sep-OTSλ plasmid - Addgene...interested in the intersection of cell signaling, metabolism, and the mighty mitochondrion. You can follow...Blog Verify Your Constructs with Colony PCR Read about Tags for Protein Purification Resources on Addgene.org...

...incorporation of a 3’-5’ exonuclease in the reaction mixture. The exonuclease removes mismatched 3’ overhangs,...scarless plasmid inserts.  Want to Learn More about DNA Assembly? Check Out Our Plasmids 101 eBook! ...to produce sgRNA for CRISPR experiments can be laborious, requiring multiple cloning steps. You can simplify...Additional Resources on the Addgene Blog Learn More about Gateway Cloning Gibson Assembly and Other Long-Homology...

...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...abundant in a sample. Whereas Southern blotting is labor intensive and requires a large amount of high-quality...DNA technologies that are now commonplace in laboratories. As scientific technologies rapidly progress...PMC4411503. Resources on the Addgene Blog Learn More about Restriction Cloning Get Tips on Analyzing DNA Sequencing...

...Figure 3 shows a histogram for a yes-no dot plot, like the one shown in Figure 1.  Figure 3: An example...interest. Created with biorender.com. The plot above is for a smeary expression and is quite different.... Additionally, you can gate off the histograms above, allowing you to visualize your data as a dot plot...

...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...customized sequence specificities." Nature communications 3 (2012): 1147. Cooke, Amy, et al. "Targeted translational...PUF Assembly Kit Below you’ll find information about what these kits contain, how they can be used, and...encoded sensors enable real-time observation of metabolite production." Proceedings of the National Academy...

...stable gRNA than when they inserted the aptamer at the 3’ end of the gRNA. Because of the success of the MS2...can be increased from 2-fold (by CRISPRainbow) to 3-fold with CRISPR-Sirius. Triple color detection With...the Addgene blog Download our CRISPR eBook Learn about fluorescent protein-based tools Read all CRISPR ...

...resulted in a roughly 6-fold increase in expression [3, Figure 2]. Note how expression increases exponentially...PMID: 18500252. PubMed Central PMCID: PMC2574934. 3. Zingg, Brian, et al. "AAV-mediated anterograde transsynaptic...diluting - if you end up concentrating your AAV above your desired titer and want to dilute it, err on...

...Knife, Genesis 34, 1–15 (2002).PubMed PMID: 12324939. 3. S. E. McGuire, G. Roman, R. L. Davis, Gene expression...Versatile Toolkit for Gene Expression in Drosophila, 3, 1–10 (2015). PubMed PMID: 21356876. 5. J. Chow, Z... use the Gal4/UAS system, and you can read more about them in review articles (2–4). You can also take...

...repression, high precision edits, and more. Figure 3: Schematic of the basic CRISPR-Cas9 mechanism, representing...double-stranded breaks caused by Cas12 create short 3’ overhangs, which leads to slightly increased efficiency...for genome-editing applications. Molecular Cell, 84(3), 463-475.e5. https://doi.org/10.1016/j.molcel.2023.12.034...the type I CRISPR system. Gene and Genome Editing, 3–4, 100013. https://doi.org/10.1016/j.ggedit.2022.100013...Class 1 is the most abundant of the two, comprising about 90% of identified CRISPR-Cas in bacteria and nearly...CRISPR systems called CasPEDIA, with information about enzyme activity, experimental conditions, and more...

...can also find Dr. Knoepfler on Twitter: @pknoepfle 3. "Social Media as a Scientist: A Very Quick Guide"...tips for using various social media platforms, and 3) helped you fit social media into your busy schedule... advantage. It wasn’t until I wrote a blog post about a research paper that explored how scientists connect...saw a request for an Addgene guest blogger post about colony PCR, I signed up immediately. I had used ...science communication skills, while also learning about new scientific developments and helping scientists... found this paper. I wrote an Addgene blog post about it and shared the paper with my lab since one of...project. I was invited to participate on a panel about “Social Media for Science Communication” at the ...

... near-infrared photosensitizer. Nature Methods,13(3), 263-268. doi:10.1038/nmeth.3735. PubMed PMID: 26808669...PMID: 18706812. PubMed Central PMCID: PMC4445427. 3. Bulina, M. E., Chudakov, D. M., Britanova, O. V.,... in culture and in vivo.  Read on to learn more about this killer illumination technique! What is a photosensitizer...within the near-infrared (NIR) spectrum (>620 nm). Above is a brief schematic of how the MG-2I-dL5** FAP-...use MG-2I-dL5** with your research? Let us know about your experiences using photosensitizers in the comments... to Receive the Fluorescent Protein eBook Learn about Small Molecule Fluorophores Resources on Addgene.org...

...MS2 RNA aptamers at the tetraloop and stem-loop 2; 3. The MS2-P65-HSF1 plasmid which expresses the activation...LS, Kampmann M, Weissman JS. Cell. 2014 Oct 23;159(3):647-61. doi: 10.1016/j.cell.2014.09.029. Epub 2014...that screen with a system you are really confident about working for most genes and individual targeting ... system, Konermann explained, was new knowledge about Cas9’s crystal structure. “I thought maybe we could...RNA we use. What we have to do now is learn more about the rules.” He said that there is no theoretical...References & Further Reading: For more information about SAM, including a tool for getting optimized guide...

...random-walk" of a budding startup, I asked Mike to outline 3 pieces that are essential to the biotech startup puzzle... anywhere. The team should initially consist of 2-3 individuals that are aligned in vision and outcomes...transitioned away from the bench and became all about finding the best product-market fit for this set...Notably, the team must have an upfront conversation about its goals and vision for the company; this will ...section! And read on for more great information about starting a biotech company. Suggested reading The...of Sample6) for taking the time to speak with us about the biotech startup process and work environment...

...segment on modulating iPSC reprogramming  Hot Plasmid 3: Regulating telomere length with TZAP. Listen to the...of our Hot Plasmids podcast series, you'll learn about new libraries for studying B. subtilis biology, ...

...to the Segement on Ouabain Selection  Hot Plasmid 3: CRISPR base editors with enhanced range and specificity...of our Hot Plasmids podcast series, you'll learn about optogenetics tools for controlling protein activity...

... Our Segment on these new AAV Vectors Hot Plasmid 3: mCyRFP1 - a new, monomeric, and cyan-excitable red...of our Hot Plasmids podcast series, you'll learn about new red fluorescent proteins, AAV tools for targeting...

... cover letter can be found on the Addgene blog.   3. Personal statement brilliance In addition to sending... Letters of recommendation wisdom  Reach out to 2-3 individuals in your professional circle to vouch for...Rose is passionate about outreach among low-income communities to educate parents about resources that can...automated filters of consideration. If you are worried about any eligibility provision, don’t guess. Simply reach...

...modified from pSC101 with copy numbers ranging from 3-100 per cell, a stabilized promoter buffered against... expression levels returned to the baseline after 3 hours with the stabilized promoter while gene expression...is useful for many metabolic engineering and synthetic biology goals. In metabolic engineering, the expression...or can interfere with the precision needed in metabolic engineering or other synthetic biology circuits...

... they can deplete endogenous protein levels for 1-3 days. There’s also a CRISPR for that! CRISPR interference...most of the other mechanisms described. Fig. 3 dTag mechanism of protein degradation. Adapted from...which can be used with this approach - read more about them on our CRISPRa blog! Fig. 1 CRISPR ... is a lot of overlap with the systems discussed above, just with more elements of control. Tet promoters...to be site-specifically knocked in. This can be labor intensive and time consuming. If your native protein...

...provide problem-solving platforms.     Fig. 3: Different workflows involved in sharing open hardware... (2022). https://doi.org/10.1038/s41592-022-01602-3... in science brings stronger networking across laboratory and disciplinary boundaries and can allow for... that are otherwise cut off from the process.   About the author: Benedict Diederich received his PhD ...

...“blind” to this very important difference (Figure 3). If you are interested in harnessing the power ... PubMed PMID: 27089539. PubMed PMCID: PMC4835065. 3. Mock, U., Hauber, I. & Fehse, B. Digital PCR to assess...advantages over mismatch nuclease assays that will be elaborated below. Digital PCR Digital PCR (dPCR) involves...type targets. Comparatively, detection limits of about 5% mutant targets have been reported for mismatch...

...no addition of nucleotides to the 5’ spacers. The 3’ ends were modified slightly with either a 1-7 base...constructs, transcript levels of PTG1 and PTG2 were 3 and 31 times higher, respectively. In rice protoplasts...with limited packaging capacity. Still confused about the various tRNA expression strategies? No worries... Addgene. If you have any questions or thoughts about this technology, leave them in the comments below...

... of conventional gene targeting is lowered to about 2-3 months. But certain models remain more difficult...directly into a zygote and obtain a knockout mouse in about 3 months. Image courtesy of Wenning Qin. Single-stranded...cleavage right before the stop codon!  Figure 3: How to design a C-terminal insertion with Easi-CRISPR... on CRISPR Mouse Models Learn about Homology Directed Repair Learn about Cas9 Ribonucleoprotein (RNP) ... a variety of species, including the standard laboratory mouse. It’s also made targeted insertions relatively... a mouse embryo to create chimeric mice. This laborious process takes ~8-10 months. With CRISPR (B), you...

...drug clinical trial, for example).   Fig. 3: Question flowchart for choosing an ANOVA/MANOVA statistical...positive is much higher than if you only look at 2 or 3 pairwise comparisons. Similar to family-wise error...for the nonstatistician: Part I. South Med J., 105(3): 126-130 (2012). 10.1097/SMJ.0b013e3182498ad5 Curran-Everett...a summary snapshot without telling you anything about outliers or data spread within those data sets. ...the rules” and which we have certain information about. The assumptions and requirements that need to be...Quantitative If your data doesn’t fit the requirements above, a non-parametric test may be the way to go, which...only performing the comparisons you really care about. Family-wise error is the probability of a false...

...recordings in primary visual cortex (V1) layer 2/3 (L2/3). B) Fluorescence from axonal boutons of neurons...Browse the IPI Glypican Collection here! Figure 3: General glypican domain structure. Glypicans are ...insert your genetic cargo of choice. After hearing about researchers' favorite features and limitations of...development and cancer. But there is still much to learn about their myriad functions and therapeutic potential...created a very quick and efficient way to learn more about gene regulation. Find the TurboCas plasmid here!...Initiative Armamentarium Collection, a major collaborative effort to develop molecular tools for studying...

...2015 Dec 31. pii: aad5725. PubMed PMID: 26721683. 3. Nelson CE, Hakim CH, Ousterout DG, Thakore PI, Moreb... some research in human embryos. Nature. 2015 Dec 3. doi:10.1038/nature.2015.18947 5. Siva K, Covello ... by zinc finger nucleases. Mol Ther. 2015a Mar;23(3):523-32. PubMed PMID: 25492562. PubMed Central PMCID...estimated that very little dystrophin correction (about 4%) is needed to see muscle improvement, with only...diseases we’d like to treat with CRISPR. As seen above, DMD can be treated with “one-size-fits-most” NHEJ-mediated.... Precision genome editing faces the challenges above and more. One chief challenge is upregulating HDR...

...Ten Simple Rules for a Successful Collaboration. PLoS Comput Biol 3:e44 . https://doi.org/10.1371/journal.pcbi...else to focus on. Use collaborative cloud apps It is a good idea to use collaborative cloud tools such as...creative collaboration in the workplace. There are many other ways to encourage collaborations remotely...meeting? No problem! You can still creatively collaborate and brainstorm! Image from Joseph Mucira. ...serve as a springboard for future discussion. Collaboration and creativity are pivotal in writing publications...Vicens et al., 2007) geared towards enhancing collaborative writing and this recent article published in...approaches/tools we have to foster creative collaboration remotely in science are endless. Let’s adapt...

... the pH change caused by storage on dry ice (Fig. 3). However, 30 seconds was not sufficient to prevent... was a substantial reduction in infectivity after 3 weeks of exposure to pH 5.1 (Potter et al., 2014)....return the sample back to the original pH. What about putting it back in the freezer? We then tested whether...

...break membranes outside of their natural context or 3) be modified to extend at different pH levels. Domesticating...Protistology 48, 290–296 (2012). PubMed PMID: 22356923. 3. Polka, J. K. & Silver, P. A. A Tunable Protein Piston... Additional Resources on the Addgene Blog Read About Other Hot Plasmid Technologies Read Other Synthetic...