...guide RNA (sgRNA), provided it meets two conditions:  1) The sequence is unique compared to the rest of the...bystander bases” if you choose this system.  Fig. 1: Cytidine deamination takes place on the free strand...temperature-controlled genome editing. Nat. Commun. 2017 81 8, 1–9 (2017).Walton, R. T., Christie, K. A., Whittaker...(but not limited to) bacteria, yeast, plants, drosophila, worms and mammals (Browse plasmids by model ...

...construct for crystallographic and biophysical studies of protein degraders. Nat Commun., 15(1), 8885. https...and is recommended for use in western blots (Figure 1). If you prefer to produce your own antibody or want...are also available from Addgene!   Figure 1: Experimental validation for Anti-V5 [IPI-SV5-Pk1]...

...animal will repeatedly nose-poke at the active hole [1, 2].    Place preference/place aversion also models... can be explained primarily in two different ways 1) your hypothesis is wrong or 2) there is some problem...follow him on Twitter @derekpsimonphd.    References 1. Stuber, Garret D., et al. "Excitatory transmission...technology is improving and may one day reach the sophistication of mouse transgenics. Everything that has been...

...sequence on either side of the desired mutation (usually 1-3 mismatched bases) is sufficient for your primers...researcher at Cornell University.       References 1. Breslauer, Kenneth J., et al. "Predicting DNA duplex... PCR followed by Restriction Fragment Length Polymorphism (RFLP) analysis (right). Introduction of restriction...of that particular site with fragment length polymorphism (RFLP) analysis. In this process, if your mutation...

...total dose between 1 x 1011 and 5 x 1011 vg per animal (i.e., 10 and 50 µL of vector at 1 x 1013 vg/mL titer... and 1 x 1012 vg*. Most systemic capsids are easy to produce in high titer, but higher doses (>1 x 1012...before using it in an application.     Figure 1: AAV toolkit for targeted gene delivery to the brain...decision tree for selecting a capsid, while Table 1 gives a brief overview of published descriptions of...Capsids page to learn more about that collection. Table 1: Systemic capsid plasmids available from Addgene ...AAV9. At lower viral titers than AAV-PHP.B (e.g., 1 x 1011 vg/mouse), AAV-PHP.eB can transduce a greater...Diverse Brain Cells. Annual Review of Neuroscience, 45(1), 447–469. https://doi.org/10.1146/annurev-neuro-111020...

...Resist the urge to say everything I allow 1 slide for every 1 minute of talk length, and then I cut a few...NatureJobs: Interviews: The All-Important Job Talk. 1. Watch other people give great talks We are not talking...Like graphing data, choosing controls, or mixing clear solutions—public speaking is skill that any scientist...

...prompted us to create Crowdfight.   Figure 1: Crowdfight is a community of scientists who believe...collaborator for a specific task, Crowdfight will: 1) Find an expert with the right skill; 2) Confirm the...volunteers helped with things like translations, bibliographic searches, etc.     Expanding collaborations...

...for locating and containing escaped flies. Nature 4:1-22. https://doi.org/4.01.2022/hafd...FLIS and Flies  Drosophila, more commonly known as fruit flies, have been an important animal model for...

...Hsp70, the heat shock releases heat shock factor 1 (HSF-1), which subsequently binds to heat shock elements...genome engineering in species like C. elegans and Drosophila. Light inducible promoters Light is another way...

...deletions, and all possible base-to-base changes.   1. Plasmids 101: Transformation, Transduction, Bacterial...Scientific Graphics by Beth Kenkel Find a round-up of tools you can use for making scientific graphics. PS:...

...post about multiplexing with Cpf1.   Figure 1: Multiplexing allows researchers to express multiple...be used in a wide range of bacteria. References 1. Maddalo, Danilo, et al. “In vivo engineering of oncogenic...other model systems, including E. coli, plants, Drosophila, and zebrafish!   Why use multiplexed gRNAs?...restriction enzyme BsaI. If you’re working in Drosophila, a two-gRNA expressing plasmid is available from...supplied on a separate plasmid. Multiplexing in Drosophila Bullock Lab Multiplex Plasmid: Two gRNAs can ...or SLIC cloning. gRNAs are expressed from two Drosophila U6 promoters. Cas9 must be supplied on a separate...efficient germline and somatic genome engineering in Drosophila.” Proceedings of the National Academy of Sciences...

...light-sensitive LOV2 domain of Avena Sativa phototropin 1 (AsLOV2). In the dark, this peptide is caged by the...iLID nano and 800nM to 47µM for iLID micro; Figure 1). With this great affinity range, these LIDs have ...Figure 2). They are all available now at Addgene. 1 Lungu et al., Chem Biol. 2012 Apr 20;19(4):507-17....Guntas et al., Proc Natl Acad Sci USA 2015 Jan 6; 112(1):112-7. doi: 10.1073/pnas. CRISPR-Cas9 optogenetic... al., BMC Molecular Biology. 2013 August 20th; 14(1). TRICK: A method for visualizing the first round...for a variety of experimental purposes including (1) single or multiple, constitutive or inducible, gene...the years. The authors optimized these systems by: 1) decreasing the number of plasmids required for each...

...Introduction to Biosensors. Essays Biochem. 2016 Jun 30;60(1):1-8. DOI: 10.1042/EBC20150001 Leopold, A. V., Shcherbakova...of the biosensors used in research are very sophisticated, but at their core they can all be boiled down...

...modified slightly with either a 1-7 base poly(U)tail in tRNA-gRNA constructs or a 1-4 base tail from the second...three strategies described in this post in the graphic below - including some simple comparisons. The...

...at a low angle (FSC) or large angle (SSC) (Figure 1). FSC values depend on the cell's size, while SSC ... or ask experienced colleagues.   Figure 1: The laser pulse illuminates the cell and is scattered...and W describe the shape of a histogram, which graphically represents the time and intensity of the cell...wondering which second measurement to choose for graphically displaying your events. You are free to choose...

...healthcare system was $35.2 billion (or $140 per $1 invested in the trial). The goal of gendered innovations...is the goal of Gendered Innovations. This project: 1) develops state-of-the-art methods of sex and gender... work to be done! Researchers need to learn sophisticated methods of sex and gender analysis. Universities...

...blotting, named for its inventor Edward M. Southern (1), can be used to detect DNA identity, size, and abundance...hours at low cost with restriction mapping (Figure 1). Though some labs may prefer to send DNA directly... serotonergic system and behavior.     References 1. Southern, Edwin Mellor. "Detection of specific sequences...simian virus 40 DNA by restriction endonuclease of Hemophilus influenzae." Proceedings of the National Academy...

...expression inducible by red light. pCph8 50552 pLtetO-1 Anhydrotetracycline Jeffrey Tabor Anhydrotetracycline... pZE31ms2 25860 MS2 coat protein binding sites Philippe Cluzel RNA localization: Contains a 2x array of...DsRed-ms2x2 26248 MS2 coat protein binding sites Philippe Cluzel RNA localization: Contains a 2x array of...Fluorescent protein, GUS, and LacZ Derivatives avaliable Philip Poole A family of promoter probe vectors incorporating...

...to do that than in the Addgene blog?  Fig. 1: One of our favorite content collections is our Help...of text. Since our guides are heavily used, our graphic designer made most of the Antibody Guide figures...communication between our content team and our graphic designer to make sure things are clear, accurate...Fig. 2: Reference image (left) provided to our graphic designer upon request that the science communicator...design (right). Immediate and immense regret of the graphic designer not shown.   However, for our blog...

...all the colors of the CRISPRainbow. Figure 1: CRISPRainbow was used to track the localization of...is available as a kit from Addgene.   References 1. Chen, Baohui, et al. “Dynamic Imaging of Genomic ...this rainbow of colors, Ma et al. performed sophisticated tracking of multiple chromosomal loci in live...

...The main differences between Cas7-11 and Cas13 are 1). non-specific RNA cleavage and 2). cell toxicity.... targeting single stranded RNA.   Table 1: Fast facts on Cas family proteins and genome editing...to detect variants such as single nucleotide polymorphisms. Fast facts – PAM requirement: none. Best ...

...expression. Brown et al., Genesis 2014 Jan;52(1):39-48. APEX2 for proteomic mapping and electron...engineering cells that lack release factor one (RF-1; Bacterial strain EcAR7) and minimizing the set of... of human mitogen-activated ERK activating kinase 1 (MEK1) with either one or two phosphoserine residues...comprised of three sets of cloning vectors (Level 0, 1, or 2) which can be utilized in three successive assembly... vectors are directionally assembled into a Level 1 vector creating a single transcriptional unit (Ex:...coding region, and terminator). Next, up to six Level 1 modules can be similarly assembled into a Level 2 ...vectors to allow for additional iterations of Level 1 assembly if necessary. Combining multiple Level 2 ...

...in a “single pot.’   Figure 1: Single-Pot Assembly: a) Graphic depiction of chromatin within a nucleus...AID system relies on a transport inhibitor response 1 (TIR1) expressed under a desired promoter with a specific...

...of an industry H-1B need to be aware of the April 1 deadline for companies to submit an application on...and non-profit organizations do not have the April 1 H-1B queue process or quota like industry, these organizations... National Interest Waiver. The three prongs are: (1) The foreign national’s proposed work endeavor must...-1B, you need to prove 3 threshold requirements: (1) that you have international recognition for your ... post may contain technical inaccuracies or typographical errors.  No action should be taken in reliance...

... Hot plasmids covered in this segment Hot Plasmid 1: CRISPR X from Michael Bassik's Lab - A new tool for...Ralser's Lab -  Use this kit to complement unused auxotrophies in Saccharomyces strains.  Listen to Our Segment...

...targeting MNase to antibody bound proteins (Figure 1). Importantly, since Protein A has weaker affinity...tissues or organs in whole animal models like Drosophila melanogaster, obtaining enough material for a...elements is controlled during development using the Drosophila wing as a model. A typical ChIP-Seq experiment...

...the brain where DA quantitation is desired (Figure 1). The probe perfuses liquid, which causes the passive...sensors in experiments The Li lab used GRABDA in drosophila, zebrafish, and mouse brain slices and freely...Demonstrate DA detection in a single neuron in vivo in Drosophila Detect DA dynamics in response to a threatening...

...into three main effector functions.  Figure 1: Fc effector functions in an immune response to a ...interaction between IgE antibodies and eosinophils, where the eosinophils release cytotoxins via degranulation...

...has error rates that range from roughly 1 per 500bp to roughly 1 per 10 million bp depending on the polymerase...recipient plasmid to insert ratio of approximately 1:3. Since the number of base pairs for each varies,...cells. For most standard cloning, you can transform 1-2μl of your ligation reaction into competent cells...analyzing the gene’s function. The process is shown graphically in the following cartoon, in which we are adding...

...crevices proteins on the cell surface. Figure 1: Comparison of common antibody, HCab, and nanobody...Humana Press, pp 133–148. https://doi.org/10.1007/978-1-62703-293-3_10 Walter J et al. (2020) Synthetic nanobodies... membranes is tricky because they have fewer hydrophilic surfaces, require detergents or lipids to remain...

...more applications.   Fig. 1 Making a Protein Tag (Abridged) (1) Locate a promising monoclonal antibody...genes: deletion mapping functional domains of Drosophila hsp 70. Embo J 3:3087–3093. https://doi.org/10.1002...

... Eric Campeau 21474 pLenti CMV V5-LUC Blast (w567-1) Firefly CMV Lentiviral expression of firefly luciferase...expression of firefly luciferase William Kaelin 21375 pHIV-Luciferase Firefly EF1α/IRES Lentiviral expression...stability and light output Sanjiv Sam Gambhir 104587 pHIV-iRFP720-E2A-Luc Firefly EF1α Lentiviral expression...

...of Contents A. pLKO.1-TRC Cloning Vector A.1 The RNAi Consortium A.2 Map of pLKO.1 A.3 Related plasmids...Order oligos compatible with pLKO.1 C. Cloning shRNA oligos into pLKO.1 C.1 Recommended materials C.2 Annealing... References H.1 Published articles H.2 Web resources I. Appendix I.1 Sequence of pLKO.1 TRC-Cloning Vector...information Back to Top A. pLKO.1-TRC Cloning Vector A.1 The RNAi Consortium The pLKO.1 cloning vector is the backbone...marker encoded in pLKO.1 allows for convenient stable selection. Figure 1 : Map of pLKO.1 containing an shRNA...puromycin should be from 1-10 μg/mL in 1 μg/mL increments. d. Label plates from 1-10 and add appropriate...Appendix I.1. Sequence of pLKO.1 TRC-Cloning Vector Click here to see the sequence of pLKO.1 TRC-cloning...

...you do just this, probably in less than a minute (1). Green Listed is a new software tool used to design...found through https://wermelinglab.com/.   References 1. Panda, Sudeepta Kumar, et al. "Green listed—a CRISPR...different academic labs, targeting mouse, human, Drosophila melanogaster, and T. gondii are available through...Central PMCID: PMC4744125. 3. Sanjana, Neville E., Ophir Shalem, and Feng Zhang. "Improved vectors and genome-wide...

...Article contributed by Susanna Bachle Figure 1: Initial steps in the CRISPR-pooled screening using...used in many cell types and organisms including Drosophila, fish, and mice, by replacing the effector with...labeling of the calcium indicator, GCaMP6f, in the Drosophila optic lobe. Image from Isaacman-Beck et al., ...

...school to a startup and back to academia. Chapter 1: How working at a startup prepared Tom for an academic...project. His team also investigates bacteria and extremophiles for biotechnology and has been directly involved...

.... Follow David on Twitter @driglar.   References: 1. Alberts, Bruce, et al. "Rescuing US biomedical research...special category, as has been the ‘labor-of-love’ philosophy of academic science to date. Advocacy opportunities...that the shifts caused to the practical and philosophical landscape of the scientific enterprise will ...University Ray Howell -Founder of Howell Communications Philip Brenner, PhD - Assistant Professor of Sociology...

...older epitope tags - only time will tell! References 1. Georgieva, Maya V., et al. "Inntags: small self-structured...and the chaperone Ydj1, both of which display sophisticated interactions with multiple other proteins. Again...

...designed to shuttle between species. Figure 1: Shuttle vectors are vectors that can function in ...the vector to survive while the auxotrophic cells die. Some auxotrophic selection markers can be used across...choose to ensure it works in your host species! Auxotrophic selection Another option is to use a different...different method of selection entirely. Auxotrophic selection relies on the host organism being unable to synthesize... example, in both S. cerevisiae and E. coli. Auxotrophic selection is especially common in yeast experiments...spontaneously develop resistance to selection toxins. Auxotrophic selection is useful for producing stable mammalian...

... genes with a single viral vector.   Fig. 1: Schematic of multiplex gene editing with DAP array... multiple genetic diseases, such as muscular dystrophies, heart disease, and type 2 diabetes, across four...multiplex genome engineering of crops or allow for sophisticated CRISPR screenings that would perturb the unprecedented...