...selection-based gene tagging approach Multiple features of C. elegans have made CRISPR/Cas9 so successful... sequences are inserted into the genome. Figure 1: Self-excising cassette schematic from Dickinson...efficient for C. elegans genome modification. Figure 2: Schematic from Dickinson et al. (2015) illustrating...may prove effective in other species or in cell culture. In addition to CRISPR/Cas9 applications, SEC selection...

... possible for added sensitivity. As reported in Nature Methods, the FRET changes of these Twitch sensor...by a secondary screen in rat hippocampal neuron cultures. The optimized Twitch sensors allowed the researchers...toxic,” Merkx explained. New tools were needed to measure them. Merkx says, while in principle adapting a...them,” Merkx said. “For us, it is one of the main measures of success – whether we have really developed ...

...serotonergic neurons (measured using a red calcium sensor) leads to the release of serotonin (measured using the ...throughput manner. She helped develop a method that measures the effects of sequence variants on abundance ...metabolism of leaves can be changed by light and temperature and leaf autofluorescence can interfere with ...

...course (read our blog post on it and to see old pictures of the lab!). By 2016, we wanted to be taken seriously...working in our laboratory! Don’t worry though, we make sure that everyone gets proper equipment and safety training...project at BosLab. Once he optimizes plant cell culture and transgenesis he wants to share his knowledge...yeast transformations! Dennis Oleksyuk, Raphael Laurenceau, and Tami Pudina have initiated a soil microbiome...

...problem or complete a project, overcoming inevitable failures and setbacks. There are many roles that actively...because they enjoy spending time outdoors and with nature. Experience in the field or in a position that ...Conservationist Documentary Director Parasitology Botanist Nature Photographer/Artist Ecologist     Computer...Include any relevant job specific ‘buzz words’ to ensure recruitment agencies and employers put your resume...

...participants could share their notes together. Figure 1: Can you draw Spongebob from these instructions...visuals, or asking questions using the polling feature or the chat box. One key thing to remember regardless...getting to know the app or virtual platform and making sure you stay connected. If possible, test out the technology...connection,” says Abitua about her workshop. “We made sure that we had a backup plan in case someone’s internet...

...actually mean? What relevance does it have on the structure of a bacteria species and how it can be used in...differences in cell wall composition. Cell wall structure Gram positive bacteria, or monoderms, have a single...the gram negative bacteria’s thinner membrane. Procedure The stain is a pretty simple process. First, flood... them red or pink. At the end of the staining procedure, gram positive bacteria are stained purple or ...

...CRISPR screening.   Using tRNAs Luckily, a fun feature of tRNA was fully unleashed by the Gao Lab’s work... for gene editing. They named this new array architecture the DAP array, short for “drive-and-process....payloads. Different from existing multiplex array architectures, including previous tRNA-gRNA arrays, that rely...Comparison of previous representative dual-gRNA architectures with DAP array   Qichen Yuan is a PhD...

..., analyzed your data, and made your tables and figures. Now it's time to write. Sometimes, it's hard to...Linux.  Mendeley and Zotero have a lot of the same features, but this chart highlights the differences between...Can Grad School podcast episode 2 "Organizing Literature" : I found out about Zotero here Dear Grad Student...blog: Tips for writing scientific manuscripts: literature searching, reading, and organizing Tips for writing...

...use agreements like MTAs.  Addgene does a lot to ensure that we can facilitate a fast and agile MTA process...material under the specified terms. This acts as the signature on any subsequent MTA for that plasmid facilitated... handled the majority of our MTA requests and signatures via an online process, though the process has...materials are requested, instead of coordinating signatures from two different institutes. Our online portal...

...and with a group who all strive for a common big-picture goal. It is worth doing some some serious pre-work...position. Am I choosing the right mentor? Make sure your advisors will be good role models and teach... career path?  If you are considering this, make sure to choose an advisor that supports both paths – ...details and many more suggestions on planning for future success by finding and joining your dream lab, ...

... conference via social media. The podcast also features an interview with Dr. Stephani Page, the founder...about a week and then unfollow those who do not capture your interest during that week.” You can also find... Media as a Scientist: A Very Quick Guide" from Nature Jobs Blog While Twitter is a popular platform for...pointers on how to use LinkedIn to research potential future employers and to arrange informational interviews...

...distribution and interactions of proteins within these structures. Enzyme-catalyzed proximity labeling (PL) uses...limitations of APEX2 and BioID. In the Ting Lab’s recent Nature Biotechnology paper, they describe TurboID and ..., the enzymes function across a much broader temperature range than BioID, which only functions optimally... the SH3 domain collection here! Teyra et al. Structure 2017. Pubmed PMID: 28890361. ...

...pandemic. Though we’ve confirmed the expected features and sequences in each plasmid, none of these constructs...will give your research more visibility.    Figure 1: Illustration of SARS-CoV-2 from Philippa Steinberg... plasmids to confirm they contain the expected features and are free of mutation.  Once the samples arrive...scientists to confirm the plasmids contain all expected features and meet our QC standards. If any concerns are...

...can affect reported titer by either method so to ensure equal animal dosing, we advise that users keep ...into codelivery efficiency at different doses. See Figure 4 in this paper for more information.   Q6 & Q7...Brain potency for each individual is reported in Figure 4B (see left for image and link for paper.)    ...and smaller CRISPR tools is an exciting area for future AAV application to epigenetic editing. This area...

...pitfalls? Here's what you need to know.   Figure 1: Management can seem a mystery at first! Image...potential. I often thought that performing a cell culture experiment myself would take less time than the...independence, while others required more guidance and structure. I quickly learned that my approach needed to ...approaches—one detail-focused and the other big-picture oriented. Their misalignment was slowing progress...

...preceded by GA or followed by TC. As shown in the figure below, a Dam methylase recognition site (underlined... diagnostics, we suggest double checking to make sure your results will not be affected by methylation... has the XbaI site blocked by methylation), the figure below illustrates how sequence visualization software...will be blocked by Dam or Dcm methylation, you can ensure this site remains unblocked by first cloning your...

...are required for NHEJ repair regardless of end structure, and dictate the major events of the pathway: ...polymerases (Pol μ and Pol λ), nucleases (Artemis), and structure-specific end cleaning enzymes (Aprataxin, Tdp2... obligatorily introduce indels. Given the end structure of the Cas9 DSB (blunt or near-blunt ends without...graduate student interested in determining how the structure of broken ends impacts how they are repaired. ...

...engineering technology. Monoculture and chemical spraying, mainstays of modern agriculture, are now synonymous... “GM” by demonstrating the current benefits and future potential of the technology to the public in an...enjoyed exploring the outdoors and experiencing nature first-hand. He studied biology at Virginia Tech...

...for vector propogation. This post, along with a future companion post on mammalian vectors, will catch... you up on the core replication and resistance features of yeast vectors and explain how they differ from...eukaryotes and thus contain complex internal cell structures similar to those of plants and animals. Unlike...Saccharomyces cerevisiae; however, we’ve also noted some features required for the replication of fission yeast,...

...possibly giving you a way to connect with them in the future. Some profiles have a blue Follow button. For less...LinkedIn page. This can include news, inclusion, culture and connections. Start your interview research ...and “plus skills” areas. Include a professional picture. It doesn’t have to be a headshot in a suit, but.... Try looking a bit over your shoulder for the picture. It tends to look like a police photo if you look...

...context. While native purification preserves RNA structure and naturally occurring complexes present in the...which for RaPID is an RNA motif of interest. See figure 1 below for details on the key components of RaPID...in breast cancer patients. By digging into the literature and analyzing data from The Cancer Genome Atlas...–protein interaction detection in living cells. Nature Methods, 15, 207-212. PubMed PMID: 29400715. PubMed...

...and the electroporated cells re-adhered to the culture plate within 4 hr of treatment. In some primary...super-resolution light microscopy, comes from his recent Nature Communications article “SRpHi ratiometric pH biosensors...to each transcription factor. They then induced cultures of these transformants with various concentrations...predicts that this level of control will enable future researchers to better simulate physiological gene...

...fluorescent in situ hybridization (FISH) and chromosome capture suffer from low resolution and can’t be used in...using all the colors of the CRISPRainbow. Figure 1: CRISPRainbow was used to track the localization...image represents the composite of fluorescent measurements from red, blue, and green channels overlaid ...with dCas9 supplied by a separate vector. Figure 2: CRISPRainbow generates 6 unique colors and a...

...plasmid, succumb to the toxicity of CcdB.   Figure 1: Image from Bernard, P., et al. showing the pKIL...still express ccdB and therefore would die. This procedure dramatically reduces the number of clones that...contain the desired insert should not grow.   Figure 2: Image from http://teachline.ls.huji.ac.il/72682...all of the players becomes ever more important to ensure that you choose the most appropriate method for...

...developing assays.  Protocol architecture The basic (very basic!) architecture of the indirect approach is...indirectly, with each protein of interest.    Figure 1: The direct approach (left) versus the indirect...best to use the direct method when possible, or ensure that the primary antibodies are different species...

...antibody you’ll want to be sure to choose the appropriate controls to be sure the staining you are seeing...produced Polyclonal antibodies are a heterogeneous mixture of many antibodies that recognize the same protein...change. If your protein might become slightly denatured during your experimental protocol, using a polyclonal...

...before me had been assigned a structure paper with two single-panel figures, one of which was a cartoon ...molecular biology class. A Cell paper. With six figures. Averaging ten panels each. (For this story, it...notebook, opened it, found my outline, read over Figure 4, took a deep breath, and, re-oriented, continued...

...Addgene is ending 2024 with big plans for our future. And while we’ve been excited to share them with...now-common business practices, like accepting e-signatures and designing a website users could easily navigate...grow accessibility alongside our repository.  “I’m sure you hear it all the time, but Addgene is a totally...institutions with standardized agreements and electronic signatures to dramatically speed up MTA approvals. In 2013...

...CARM1 methyltransferase. This attendant signaling feature afforded by coactivator complexes suggests that...the NURSA website, the new version of which will feature a simplified user interface, expanded numbers of...associated with identifying plasmids in the research literature and acquiring them in a timely manner. Addgene...accordingly, the new version of the NURSA website will feature direct links to listings of available plasmids ...

..., 2013). Two important features of this early system are its regulatable nature and its ability to multiplex...occurred. This powerful negative selection system ensures high editing efficiency; the only non-edited cells... pTargetF and pCas plasmids can be subsequently cured from the bacteria. Although developed in E. coli...Resources on the Addgene Blog Check out our CRISPR Featured Topic Page Learn more about Cpf1 Find out about...

...inheritance: 1. Genotype your parental mice In order to ensure that your genetic modification will be passed along...genotype all of your F1 mice, as well as progeny from future generations. 4. Continue breeding your colony You...they are likely to stay on separate chromosomes in future generations as well, a concept known as linkage...expression of Cre, loxP, and your gene of interest. To ensure that the effects you see in your experiment are...

...et al. "cGAL, a temperature-robust GAL4–UAS system for Caenorhabditis elegans." Nature methods 14.2 (2017... detailed information about C. elegans 3’UTRs structures, alternative polyadenylation for all protein ... novel findings. These results are normally one figure and can be negative and/or even replicated studies...

...those which you’ve already fluorescently labeled. Figure 1: Using Split-BioID to Identify Protein Protein...that give the strongest biotinylation signal, as measured via Western blot, and that result in proper localization...these two bait proteins stop interacting. This feature is particularly useful for in-depth characterization... of spatiotemporally defined protein complexes. Nature communications. PubMed PMID: 28585547. PubMed Central...

...infections. She is particularly passionate about HIV cure research and science communication.     References...to the central and peripheral nervous systems." Nature Neuroscience 20.8 (2017): 1172-1179. PubMed PMID...affinity-purified receptor-targeted viral vectors." Nature communications 6 (2015): ncomms7246. PubMed PMID... the Addgene Blog Check out our Viral Vectors Featured Topic Page Learn about CRISPR-AAV vectors Learn...

...engineered CRISPR-Cas9 complex. Nature 517:583–588 . https://doi.org/10.1038/nature14136 Perez-Pinera P, Kocak ...acetyltransferase domain capable of rearranging chromatin structure. This method has shown higher activation levels...C, Hsu PD, Habib N, Gootenberg JS, Nishimasu H, Nureki O, Zhang F (2014) Genome-scale transcriptional ...

...an anti-CRISPR protein. Nature 546:436–439 . https://doi.org/10.1038/nature22377 Harrington LB, Doxzen ... species of bacteria.  A note on anti-CRISPR nomenclature Acr family proteins are named for the type of... DNA cleavage (Harrington et al., 2017). Figure 1: Anti-CRISPR proteins (Acr's) can block CRISPR...

...gene delivery, but it has a drawback: how do you ensure your gene goes where you want it to? Knowing that...transcriptionally active in retinal cell types in mice. Figure 1: The four strategies used to design synthetic...AAVs would express Cre-mCherry and GFP.   Figure 2: Synthetic promoters can be paired for targeted...promoter AAVs in mice, non-human primates and humans. Nature Neuroscience, 22, 1345-1356. https://doi.org/10.1038...

...efficiently and accurately in a “single pot.’   Figure 1: Single-Pot Assembly: a) Graphic depiction of... sensor modules targeting a specific chromatin feature (e.g. a methylated histone such as H3K27me3), a...neuron-astrocyte, and cancer-stromal cell pairs.   Figure 2: Schematic of the interaction between the sender...quantification of this depletion using imaging-based measurements (Ashley et al., 2021).   Schematic showing...

...the impacts of neural circuits on behavior. Figure 1: An overview of the chemogenetics system. ...characterize the compound, or compounds, you choose to ensure reliable results (Goutaudier et al., 2019). Using...Yang X, Stone O, Roth BL, Jin J (2015) The First Structure–Activity Relationship Studies for Designer Receptors...designed Gi-coupled receptor in transgenic mice. Nature Biotechnology 17:165–169 . https://doi.org/10.1038...

...animal’s skull using dental cement (a similar procedure as implanting a guide cannula). A fiber optic ...optogenetics but may become the standard in the future. Now that your animals are all wired up and expressing...long-term optogenetic manipulation of neural circuits. Nature protocols. 2012;7(1):12-23. doi: 10.1038/nprot.2011.413...Neuroscience research. 2011;70(1):124-7. doi: 10.1016/j.neures.2011.01.007. PubMed PMID: 21238511. 6. Jeong, ...

... explain the thinking behind the tool, its key features, how it works, and how to use it. Background on...frequency determination (CFD score) is used to measure specificity of a guide to the target. The CFD score...Optimal mutation in-depth One the most sophisticated features of CrispyCrunch is how it mutates guide sequences...more detailed information on using CrispyCrunch, be sure to check out the How To Guide. Conclusion CrispyCrunch...

...graduate school who were at AstraZeneca and could assure me that they were having a positive experience,... years. This time limit can feel like a lot of pressure. However, it means that your manager (and you)... choose. If publishing is important to you, make sure that your project will not involve proprietary compounds...reviewed by an internal committee before submission to ensure that it does not divulge any propriety information...

... 25°C. At low temperatures like 18°C, flies take longer to develop. At high temperatures like 30°C, flies...drift away with each passing generation.  Another feature of balancer chromosomes is that they produce dominant... during meiosis. This can sometimes be a handy feature when setting up genetic crosses. More importantly...

...been used to track protein localization, cell structure, intracellular trafficking, and protein turnover...plasmids to fluorescently tag markers of cellular structures in mammalian cells. These plasmids use fluorescent...reversibly degraded after the addition of auxin to the culture medium. Any number of regulatory sequences, including...“A Comprehensive Toolbox for Genome Editing in Cultured Drosophila melanogaster Cells.” G3 (Bethesda) ...

... new experimental species. Discovering ways to culture, propagate, transform, store, and share all of ... "for development of tools, approaches, and infrastructure necessary for direct tests of cause and effect...was excited to be invited to attend. My job is to ensure that, in general, these labs all include materials...from Old Faithful in 60 °C soils. Dendrobatidae (Lauren O’Connell)  Poison frogs are great for studying...

...the same time, this project gives Snapgene great exposure to the community of biological scientists who ...institutions personally on conference calls and make sure to maintain these relationships by keeping in touch...an internal to an external connection is to make sure you keep open lines of communication with colleagues...interesting events, articles and newsletters I make sure to share them with a short note - this creates a...

...stages (i.e. postdoc level) and can include the pressure to publish in high-impact journals, and the requirement...with a custom-made reagent to manipulate cells (manufactured by one of the larger biotech companies), and...company, less exposed to the fast dynamics and pressure which are often found in sales or lab-based roles...name a few). Depending on the particular company structure you may be able to move into one of these departments...

...sent to the AbuGoot lab for testing, they weren’t sure what they would find… but it was safe to say the...dual activities: processing pre-CRISPR RNA into mature CRISPR RNA and cleaving RNA at positions defined.... But they weren’t done engineering quite yet – surely, they thought, the integration rate could be improved...with the single-protein CRISPR effector Cas7-11. Nature. 2021 Sep;597(7878):720-725. doi: 10.1038/s41586...

... than studies in other model organisms or cell culture. For example, Scott Lowe’s lab utilized mice to...double-stranded RNA in Caenorhabditis elegans." nature391.6669 (1998): 806. PubMed PMID: 9486653. Hesselson... and its relationship to the human genome." Nature496.7446 (2013): 498. PubMed PMID: 23594743. PubMed ...organism for studying somatic mutations and cancer." Nature Education 1.1 (2008): 183. Zuber, Johannes, et ...