...environments such as live cells and tissues (Figure 1). These flexible strategies give you the best of both...majority of in-cell labeling strategies have two parts: (1) a genetically encoded “tag” expressed as a fusion...Follow him on twitter @rhodamine110.   References 1. Liu, Zhe, Luke D. Lavis, and Eric Betzig. "Imaging...individual molecules. Poor photon budgets are a widespread issue with fluorescent proteins—even the thriftiest...student searching couch cushions for spare change, desperate to extract a few more photons from a sample. Chemical... the complexity of the system. A particularly desirable property for small-molecule labeling strategies...size, brightness, and cell permeability. In past decades, further refinements to this established dye structure...

...-public settings. Ask over coffee.  Have regular 1:1 meetings with team members (don’t become known for...communication between all members of a team. As described here in a 2012 Intuit blog post, a study coming...

...a rate of up to 1 per 500-1000 bp. In comparison, the DNA replication error rate is 1 per 109 bp. Unlike...common 30bp overlap sequences (Sites #1-5). Each overlap site encodes a unique restriction enzyme site and...Transcriptional Output-TANGO (PRESTO-TANGO) to screen the NCC-1 library of approved drugs against the entire kit; ...First, GMAP-compatible backbones for lentivirus LV 1-5 and retrovirus RV 2-5 were constructed. The authors...is multiplex automated genome engineering (MAGE) (1). MAGE uses recombineering (2) to simultaneously incorporate...of GFP (SEP) by a 9 amino-acid linker (see Figure 1). The key to the biosensor lies in pH: DsRed is relatively...inactivate SEP and leave only DsRed to fluoresce (1). With variants that can be targeted to specific cellular...

...optimized for expression in E. coli: acoustic gene 1 (arg1) and acoustic gene 2 (arg2). The key difference... plasmids are available from Addgene! References 1 Bourdeau, R.W., Lee-Gosselin, A., Lakshmanan, A., ...have hollow interiors and a protein shell that excludes water but is gas permeable. These vesicles are...sequentially exposed to acoustic pulses with high enough amplitudes to first collapse arg2 and then arg1 vesicles...

.... pH and DNases affect DNA stability Figure 1: So many choices! What to do? The main factors ...your downstream experiment. TE (10 mM Tris-HCl,1 mM EDTA, pH 8.0) buffer is the best buffer for preserving... favors the breakage of the hydrogen bonds, destabilizing the molecule and favoring the separation of ...nuclease free water is a great option because it provides a great versatility on how to use your plasmid...

...while the paid version provides more tools for plasmid mapping and design (Figure 1). Benchling: While you...Davis from the University of Utah.   Figure 1: A plasmid map created in SnapGene of Blue Flame plasmid...primer design Design primers for Goldengate cloning   Design primers for In-Fusion Cloning Design primers...academic users. Serial Cloner: Free desktop-based software for plasmid design and mapping. ApE (A plasmid Editor...Primer design tools Many of the above-listed plasmid mapping tools have built-in primer design tools, ...stand-alone sites for general primer design, as well as primer design for different cloning applications...characteristics. NEB Data-optimized Assembly Design (DAD): This tool designs overhang sets for high complexity Golden...

...B. Gurumurthy, and Masato Ohtsuka. Nat Protoc. 13(1) (2018):195-215. PubMed PMID: 29266098 Creating knock-in..., with protocols using dsDNA templates achieving ~1-10% efficiency. Easi-CRISPR uses a long ssDNA template...working with CRISPR than just designing a repair template that contains your desired edit, and I want to make...say about CRISPOR, a tool to help you design, pick, and close guides for Cas9 and Cpf1. This tool supports...The blog post I mentioned, John Doench’s How to Design your gRNA for CRISPR Genome Editing, gets over ...these numbers show that this basic concept of gRNA design is still challenging for researchers using CRISPR..., and that standardizing and improving the gRNA design process and rules for various applications is necessary...

...takes a different approach, randomly removing 1-20 nucleotides from the cleavage site specified by the gDNA...About NgAgo User's First Impressions Here References 1. Gao, Feng, Xiao Z. Shen, Feng Jiang, Yongqiang Wu...DNA guides NgAgo uses 5’ phosphorylated DNA guides (so called “gDNAs”) rather than the RNA guides employed...24 base DNA guides may be more user-friendly, since they can be ordered as oligonucleotides and should ...gDNA guides will also need to be transfected into the cells of interest. In contrast, RNA guides must ...important role in RNA interference, binding RNA guides to cleave foreign RNA. Argonaute proteins are also...cleave DNA in vitro, directed by phosphorylated DNA guides. Unfortunately, Swarts et al. were unable to show...

...organoid model (Sun, et al. 2021).  Figure 1: Example schematic from the Organoid EasyTag system...without altering that of the internal control (Fig 1). Because this method relies on the fluorescence ratio...CRISPR inhibition (CRISPRi). A) Lentiviral vector design. B) Strategy for leak-free transcriptional control...Organoid EasyTag workflow combines thoughtfully designed plasmid components with Fluorescence Activated... heavy optimization. Claudia Vickers’s lab has designed novel SL fluorescent biosensors by integrating... rather than on the fluorescence intensity, it provides a direct and quantitative detection of SLs. The...biosensors could have a major economic impact.   Design and mechanism of action of the receptor-based SL...

...microscopic resolution for studying colloidal gold (1).  The method was based on using a thin plane (sheet...Siedentopf, and R,. Zsigmondy.  (1902) Ann. Phys.  315, 1 – 39.
 A. H. Voie, D. H. Burns, F. A. Spelman. (1993...microscopy/en_de/products/imaging-systems/lightsheet-z-1.html ...After this initial application, Stelzer’s group described the single-plane or selective-plane illumination...and acquire images of a sample from two opposing sides and merge the images, such as in the Ultramicroscope...

...Recently I learned that Addgene’s pLKO.1 cloning protocol is viewed around 3,000 times each month. I ...on the Addgene website in December 2006, the pLKO.1-TRC cloning vector (deposited by David Root of the...would the thousands of other scientists using pLKO.1 variants learn about those updates? This is a problem...Protocols OpenWetWare.org (OWW) is a wiki that provides a community for biologists and bioengineers to...the site. Like Labguru, Quartzy offers software designed with scientists workflow in mind and can offer...

...determined LY6A, also known as stem cell antigen-1 (SCA-1), is the receptor used by PHP.B to reach the CNS...William L. Stanford. "Concise review: stem cell antigen‐1: expression, function, and enigma." Stem cells 25.6... a receptor for PHP.B. Read on to learn how the design of PHP.B may have contributed to this mouse-specific...Cre-dependent selection yields AAV variants for widespread gene transfer to the adult brain." Nature biotechnology...

...sarcoma-associated herpesvirus.       References 1. Snapp, Erik. "Design and use of fluorescent fusion proteins ...experiments or even negatively impact cells (see Figure 1). When behaving properly, FPs enable researchers ... disulfide bonds in the secretory pathway (Figure 1) [4,6–8]. Additionally, FPs that encode N-glycosylation... live cells in real time. Previously, we have described a number of practical considerations that should...

... spread out onto the plate. We test the (capped) 1:1 agarose/DMEM solution against a part of the body ...a 60 mm dish ready for picking is shown in figure 1.   Find Additional Useful Protocols on Addgene's Protocol...investigators to identify specific clones with desirable properties, either initially, or using secondary...

...everything I allow 1 slide for every 1 minute of talk length, and then I cut a few more slides. Do not run over...NatureJobs: Interviews: The All-Important Job Talk. 1. Watch other people give great talks We are not talking...suspense. Notice the simplicity of their slides and how the slides complement the spoken message. Learn from...speak to start creating slides or thinking about content. Create a Google.slides or Powerpoint file right...how these speakers talk without looking at their slides or notes. Listen to their stories and narratives...listeners to be thinking “What happens next?”  Describe how you pushed to a solution or overcame obstacles...term, but are worried that this might sound condescending, consider explaining the term on a slide but...

...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...vectors and design your sgRNA targeting sequence using one of the many freely available sgRNA design programs...many cases several exons’ worth of DNA. Any ∼20 nucleotides of DNA can serve as the target for Cas9’s single...Cas9 localizes the deaminase to where the edit is desired, and it performs the programmed edit within the...flexibility. A consideration with this tool is that the desired edit will be performed on all relevant bases within...

...than control, 1; very dark). Brighter fluorescence indicates a more successful gRNA design. Adapted from...In vitro screening for the best gRNA Figure 1: Validation of double strand break (DSB) mediated ...and greatest in scientific content.      References 1. Cho, Seung Woo, et al. "Analysis of off-target effects... screen them for your desired edit. Good news! Remember the primers you designed to generate your pCAG-EGXXFP...CRISPR/Cas9 system floating around, and many pre-designed plasmids containing these variants ready for purchase...that do it for you. One such option is the CRISPR Design tool supplied by the Zhang lab. This simple website... and cons of using many different types of gRNA design software. When you are happy with your selection...

... to as ssODNs (oligodeoxynucleotides)) are commonly used for smaller modifications (~1-50 base pairs)....Easi-CRISPR was shown to increasing editing efficiency from 1-10% with dsDNA to 25-50% with ssODNs in mouse models...ribonucleoproteins. Quadros RM, et al. Genome Biol. 18(1); (2017). PubMed.  Additional Resources on the Addgene...your desired edit are all you need to generate a knock-in.   General considerations for designing a repair..., and ultraviolet light. In this post, we will describe the mechanism of homology directed repair, one...increase the frequency of edits, researchers have designed self-cleaving plasmids that liberate the targeting...information on HDR and CRISPR. Our CRISPR 101 series was designed to help explain the basic principles driving CRISPRs...

...catalog had grown to 879 viral catalog items (Figure 1). The service caught on quickly in the scientific ...over 100,000 viral vector preps!   Figure 1: A) Cumulative AAV preps available in the Addgene ...Neuromethods, 195, 77–101. https://doi.org/10.1007/978-1-0716-2918-5_5 Haery, L., Deverman, B. E., Matho, K...site, and titer. Addgene’s Viral Vector Service Besides being complex to use, viral vectors are also difficult...which include calcium and voltage sensors, are the broadest category of viral vector tools, which may help... for vectors in the “controls” category, which includes things like viral vectors that deliver GFP. ...most requested serotypes (Figure 4), our catalog includes vectors in a variety of serotypes, including the...

...your budget, you should take the following steps: 1. Define your timeline Start with your thesis in mind...these decisions in the following order of importance: 1. First make decisions based on experimental timelines... several factors aren’t taken into consideration. 1. Always focus on the science When negotiating finances...Wilpitz founder and lead consutant at Experiment Designs Consulting, Inc. “How much do you need [for your...necessary resources. This is why at Experimental Designs Consulting (EDC), we instead have our clients focus...the institution/funding source. Major equipment includes large centrifuges, sequencers and analyzers, and... as many of these tools as possible, this even includes clocks, timers, and phones.  As for reagents and...