...building it! Table 1 has a list of Fragmid destination vectors currently available. Table 1: Fragmid destination...components on the Fragmid website.   Figure 1: (Top) Schematic overview and timeline of the Golden... CRISPR vector the Fragmid way!    Figure 1: Selecting your vector type on the Fragmid website...components available will depend on your answers in step 1, so you’ll only be able to select components that ...Gate cloning, perform an exonuclease cleanup, transform, plate, and pick. In short, the Fragmid system...AAV pRDA_889 (ITRs from AAV2) Mammalian Transposon pRDA_757 (Piggybac; constitutive expression...   Drosophilia Plasmid pRDB_053 (for transgenic flies; gypsy insulator sequences; white + selectable...

...while its nucleolytic activity is eliminated (Fig. 1) (1, 3). The gRNA can be further modified by incorporation... Podcast Interview with Alex Chavez Figure 1: Schematic of native (nuclease-competent) Cas9 interacting...which Cas9 protein is directed to which target site (1). Although this is possible, this strategy suffers...sequence or regulating gene expression.     References 1. Esvelt, Kevin M., et al. "Orthogonal Cas9 proteins...which cleaves one strand of DNA upon binding the protospacer. The resulting double strand break (DSB) stimulates...two catalytic domains, HNH and RuvC, cleave DNA strands complementary and non-complementary to the gRNA... binds the target site, but does not cut either strand of DNA. Traditional Cas9 regulators In addition...

...preventing your primary antibody from binding (Fig.1). The level of cross-linking that occurs depends on...proteins, which could disrupt your target epitope (Fig. 1). So, while organic solvents may improve antibody ...thing depending on your experiment.   Figure 1: Choice of fixation method impacts antibody binding...New York, pp 299–311. https://doi.org/10.1007/978-1-4939-8935-5_26  Rolls G (2022) Process of Tissue Fixation...www.leicabiosystems.com/us/knowledge-pathway/fixation-and-fixatives-1-the-process-of-fixation-and-the-nature-of-fixatives...and membrane integrity (Fig. 2). For example, saponin interacts with cholesterol in cell membranes and...can lose membrane-associated proteins, but it is better at permeabilizing organellar membranes. Created...

...used by Gao et al, 2016 (1). View the Complete and Up-to-Date Results of Pooran's NgAgo Survey The take-...the genome, which seem resistant to Cas9 cleavage (1). Both the CRISPR-Cas9 and NgAgo genome engineering...it was first published in Nat Biotech in May 2016 (1), NgAgo has received much attention and the plasmid...Survey Survey results As of August 1, 2016, a total of 165 researchers responded to the survey. When asked... dreams of a sunny day in Edinburgh.   References 1. Gao, Feng, et al. "DNA-guided genome editing using...respondents (transfection method, amount of each component per transfection etc.). Take Pooran's NgAgo Survey...Doudna Lab Many thanks to our Guest Blogger Pooran Dewari! Pooran Dewari is a postdoc at the MRC Centre for...

...). Analysis of transduction efficiency, tropism and axonal transport of AAV serotypes 1, 2, 5, 6, 8 and...Facilitate Efficient Binding and Transduction by Adeno-Associated Virus Types 1 and 6. Journal of Virology,...them to target particular cell types. Figure 1: Diagram of AAV components. Created with BioRender.com...depends on your choice of promoter/enhancer. Table 1: AAV capsids and receptors. Capsid Primary receptor...required)2 Human fibroblast growth factor receptor 1 (FGFR1)3 αVβ5 and α5β1 integrins3 Hepatocyte growth...structure of AAV2, so they are formally called rAAV2/1, rAAV2/2, and so on, although we often use abbreviated... N.J.), 807, 141–157. https://doi.org/10.1007/978-1-61779-370-7_6 Tabebordbar, M., Lagerborg, K. A., Stanton...

...pitfalls? Here's what you need to know.   Figure 1: Management can seem a mystery at first! Image used...Understanding the transition While a PhD equips you with exceptional technical skills, transitioning into management...   Strategies for a successful transition To successfully transition into a managerial role, it's essential... most pivotal decisions many PhDs face when transitioning from academia to industry is whether to stay...career trajectories within the industry, but the transition wasn’t easy - I had to reinvent myself and redefine...d like to share below. Common pitfalls in the transition (what I wish I knew before experimenting on myself...being a great scientist doesn’t automatically translate to being an effective manager. Mismanaging (no...

...reactions. (Need a refresher? The five rules are: (1) don’t use the same overhang twice; (2) avoid palindromes...Gate) pot (Pryor et al., 2022).     Figure 1: Workflow for high complexity Golden Gate Assemblies...different enzymes and cycling conditions (see Table 1).   Enzyme Temperature Incubation time/cycling...cycling Overhang length T4 DNA ligase 25 °C 1 hour 3-base or 4-base T4 DNA ligase 25 °C ...hours 3-base or 4-base T4 DNA ligase 37 °C 1 hour 3-base or 4-base T4 DNA ligase 37 °C ...Ligase MM 37-16 °C cycling 3-base Table 1: Enzyme and cycling conditions available in DAD tools...up to 10-12 fragments (Pryor et al., 2020), and randomly selected non-palindromic pairs allows for high-fidelity...

...premature chain termination 1-10 ug/mL Zeocin Sh bla HeLa, NIH3T3, CHO, COS-1, 293HEK, Jurkat T cells Complexes...  HeLa, NIH3T3, CHO, COS-1, 293HEK Inhibits termination step of translation 2-10 ug/mL G418/Geneticin...expressing the Epstein–Barr virus (EBV) nuclear antigen 1 (EBNA1) or the SV40 large-T antigen (293E or 293T ... does not guarantee 100% transfection efficiency. Stable transfection A stable transfection is used to...process called transfection. Transfection is somewhat comparable to bacterial transformation (the introduction...the transfected plasmid to be expressed transiently. Since the DNA introduced in the transfection process...the genome to form a stable transfection. Unlike bacterial transformation, scientsts do not "select" for...

...here at Addgene – 500,000 plasmids shipped! That's a 1/2 million plasmids sent to happy scientists around...start out at our Cambridge office – previously at 1 Kendall Square, but newly relocated to 75 Sidney Street...Have a question? Email: [email protected] Call: +1 (617) 225-9000         ...Addgene's lab. We ship individual plasmids as transformed bacteria in stab culture format. Our lab team... and collaborations with institutions and tech transfer offices that facilitate MTAs. The great feedback...

... advanced and finally, to expert.   Figure 1. Visual representation of common injection routes ...back or hind legs Gauge: 25–27 Volume: < 2–3 ml (~1 ml per site) Skill required: beginner Intradermal ...work. Good luck with your in vivo experiments! Table 1: Summary of common injection routes in mice. Injection...hind legs; between skin and muscle 25–27 < 2–3 mL (~1 mL per site) Beginner Intradermal Anesthesia Between...resources on the Addgene blog Mouse Modeling Part 1 Mouse Modeling Part 2 Five Popular Model Organisms... 0.07 ml Skill required: advanced Intranasal instillation Intranasal instillation is very similar to its...behind the eye. IV injections are used for a broad range of substances, as it circulate what is injected ...

...system defined three components required for translation (Figure 1): A tRNA that recognizes a specific ‘open...deletion of release factor 1, the protein responsible for terminating translation in response to the presence...cellular proteins are modified through phosphorylation (1). However, the expansion of studies on protein phosphorylation... open reading frame (ORF) of interest (Figure 2). 1. The phosphoserine OTS The backbone of the phosphoserine...Rinehart lab: pCRT7 NT TOPO E17TAG GFP and pGEX-6P-1. The pCRT7 NT TOPO vector contains a mutant pSer-containing... can follow her on Twitter @nieminm.   References 1. Cohen, Philip. "The origins of protein phosphorylation...one of the most inclusive databases of post-translational modifications, identifies a staggering ~250,...

...into more specialized cells with specific functions [1]. In many tissues, stem cells serve to replenish/replace...damaged cells that no longer function adequately [1]. Stem cells’ ability to differentiate into multiple...://www.linkedin.com/in/kmukherjeephd/. References 1. Dittrich, R., M.W. Beckmann, and W. Wurfel, Non-embryo-destructive...The potential of muscle stem cells. Dev Cell, 2001. 1(3): p. 333-42. PubMed PMID: 11702945. 6. Cowan, C....preimplantation genetic diagnosis. Hum Reprod, 2008. 23(1): p. 46-53. PubMed PMID: 17989069. 8. Takahashi, K...during ESC isolation. Low Chances of post-transplantation rejection when used therapeutically High Low...similar functional utility, iPSCs bypass the transplantation compatibility issues and ethical concerns of...

... successful and meaningful career in the grants profession: 1. Build on your existing skills & passion...Associates, Inc., a national grant consulting firm, and Smartegrants, a grant professional development firm... the field of grants. What is a grant professional?  In the past 5-10 years, the Grants Profession has...readiness, identify grant opportunities, secure grant awards, and manage grant projects and funds. You will... for grants, the amount of grant funds available and the increasing regulatory process for grants has ...become a grant professional? Employment opportunities for those with expertise in grant writing, grant management...as a Grant Professional to mentor and coach you. There are many grant consultants who offer grant training...

...the Gal4/UAS system is a transcription activation system co-opted from yeast (1). It is a Drosophila geneticist...still reads “#” as a “pound symbol”. References 1. A. H. Brand, N. Perrimon, Targeted gene expression as a...Elliott, A. H. Brand, The GAL4 System : A Versatile Toolkit for Gene Expression in Drosophila, 3, 1–10 (2015... A Fly Geneticist’s Swiss Army Knife, Genesis 34, 1–15 (2002).PubMed PMID: 12324939. 3. S. E. McGuire,...workhorse to turn genes on or off. Gal4 is a transcriptional activator that binds to UAS enhancer sequences...sequences found in DNA. It then recruits transcription machinery to the site to induce gene expression. Thus...Lai, T. Lee, Genetic mosaic with dual binary transcriptional systems in Drosophila., Nat. Neurosci. 9, 703...

... arms. Nat Chem Biol., 308(20): 1-9 (2020). 10.1038/s41589-019-0432-1 Mehdi Banan. Recent advances in ...knock-ins by HDR.   Fig. 1: Early steps in repair of a DNA double strand break by homologous recombination...traditionally have larger homology arms in the 500 bp to 1 kb range, however, shorter homology tracts have recently...repair of DNA double strand breaks. J Biol Chem., 293(27): 10524-10535 (2018). 1 10.1074/jbc.TM118.000372...(single vs. double stranded) all depends on the size of the knock-in. Single strand oligonucleotide donors...knock-out. The goal of a knock-out is to disrupt DNA translation by generating a frameshift mutation. The majority...pathway cells already have for repairing DNA double strand breaks – (HR). HR is the dominant homology-directed...

...evaluate all of the pegRNAs in one experiment (Figure 1). Through this method, we found key determinants of...diverse edit types and cell contexts. Figure 1: High-throughput screening of pegRNA efficiency and...Article 3. https://doi.org/10.1038/s41576-022-00541-1 Kim, H. K., Kim, Y., Lee, S., Min, S., Bae, J. Y.,...DNA repair determinants. Nature Biotechnology 2023, 1–11. https://doi.org/10.1038/s41587-023-01678-y Li,...editing systems in multiple cell types. Cell, 186, 1–17. https://doi.org/10.1016/j.cell.2023.03.034 Resources..., and deletions) without introducing DNA double-strand breaks (Anzalone et al., 2019; Chen & Liu, 2023...typically a Cas9 H840A nickase fused to a reverse transcriptase) together with a prime editing guide RNA (pegRNA...

...the sample (by less than 1%) and recovered after the magnet was removed (Figure 1). At first, Andrew and...Image reproduced from Frank Hayward et al. 2024 under CC-BY license.   Video 1: Fluorescence movie ...since the initial report in 2023.)     Figure 1: Fluorescence intensity of EGFP-FlavinTag in E. coli...that could be expressed and purified. Although a ~1% change in signal might not make a very powerful tool...tool that responds strongly to magnetic fields (Frank Hayward et al., 2024). They deposited their plasmid...proteins. Motivated by curiosity Although a wide range of fluorescent proteins and optogenetic tools exist...when a small magnet was passed over the sample (Frank Hayward et al., 2024). The fluorescence intensity...

...simple two-step, 2 hr protocol prior to transformation (Figure 1). As this approach requires no special...allow immediate co-transformation with linearized vector backbone. DpnI digestion - 1 μl DpnI is added...confirm primer design.  Use minimal template DNA Use 1 ng of PCR template DNA to minimize false positive ... by PCR, so we usually add DMSO (3%) and Betaine (1 M) to the PCR mix. When PCR is not possible Some plasmids...Twitter @jakefwatson and @JGarciaNafria.  References 1. Jones, Douglas H., and Bruce H. Howard. "A rapid ...false positive colonies. Transformation - the linear fragments are transformed into E. coli and plasmid...assembly of a circular plasmid in vitro, before transforming it into E. coli for propagation. However, while...

... class 1 systems. A putative new class 2 nuclease, Cpf1 (CRISPR from Prevotella and Francisella), was ...guide for a refresher on CRISPR/Cas9.  Figure 1: Cpf1 is a single RNA-guided endonuclease of a class...that Cpf1 can tolerate mismatches at gRNA positions 1, 8, 9, and 19-23. Accordingly, the 3’ end of the gRNA...Most gRNAs directed low-frequency Cpf1 cleavage at 1-12 off-target sites; in contrast, SpCas9 may cleave...the diversity of CRISPR-based systems. References 1. Zetsche, Bernd, et al. "Cpf1 is a single RNA-guided... Cas9’s shortcomings - namely its blunt double stranded cleavage and G-rich PAM requirement. Zetsche et...one to characterize the Cpf1 nucleases. Using Francisella Cpf1 (FnCpf1), they employed an E. coli plasmid...

...Cre-expressing AAV-5 into the DRN of a transgenic mouse harbouring a Cre-dependent IL-1 beta overexpression cassette...production of the pro-inflammatory cytokine interleukin IL-1 beta in the dorsal raphe nucleus (DRN), a region thought...Cre in these neurons turns on overexpression of IL-1 beta. They tested the mice in a series of behavioral...risky), or a closed arm (safe). Mice with higher IL-1 beta production spent more time in the open arm and... part of the apparatus, it turned out that the IL-1 beta overexpressing mice spent more time in the risky...project to the NAc, where enhanced glutamate transmission leads to locomotion sensitization. (for review... Channelrhodopsin 2 (ChR2) reduced glutamate transmission from the mPFC projections to the NAc and decreased...