...mammalian evolution. Nature 428, 493–521 (2004). https://doi.org/10.1038/nature02426 Iannaccone, P. M., ...processes and developments. Model organisms often feature genetic, physiological, or developmental characteristics...laboratory settings. One of its most attractive features is its stability and easily obtainable growing...axolotl has the ability to regenerate complex structures such as limbs, the spinal cord, and internal ...disease research. One of their most attractive features are their eggs. Frog eggs are large, opaque, and...genomics study in bacteria with superior performance. Nature Communications, 9(1). https://doi.org/10.1038/s41467...

...get several interviews but no job offers, is a failure to clearly articulate one's talents and ideas. ...Have a competitive CV You don't necessarily need a Nature/Cell/Science paper. You do need multiple first/...techniques. You DO need to be prepared to know the literature of your field and all of the caveats and alternatives...misfolded proteins is essential for human health. Failure to do so is implicated in several human diseases...much easier to focus on the questions when you’re secure in the knowledge that you have given everyone a...you want to convey for each section or aim. Make sure those get priority. The fine details for aficionados... is the task. Do your homework. Learn what your future potential colleagues study. Think about how you...

...four-stranded branched structures that form when elongation of the invasive strand “captures” and synthesizes...genome instability and contributes to cancer, premature aging, and other harmful processes. The most dangerous...strand; this results in the formation of a DNA structure referred to as the displacement loop (D loop)....and the resulting mutations after an HDR event, ensure the gRNA or PAM site is disrupted in the donor....engineering using the CRISPR-Cas9 system. Ran FA, et al. Nature Protocols Nov;8(11):2281-308; (2013). PubMed. ...Resources on the Addgene Blog Check out our CRISPR Featured Topic Page Learn about Non-Homologous End Joining...

... each requires selection. These two steps are (Figure 1): The initial plasmid integration step (the ...antibiotic resistance marker from the vector (abR, Figure 1). The resolution step (“double crossover”) which...for the loss of a specific phenotype (using sacB, Figure 1, further discussed below).  Counter-selectable...frequently requires considerable optimization to ensure strong counter-selection. Anecdotally, there is... of two visible blue or magenta chromoproteins (Figure 2) (Liljeruhm et al., 2018). This system is a nice...aeruginosa genome with two-step allelic exchange." Nature protocols 10.11 (2015): 1820. PubMed PMID: 26492139...of bacterial genomes using CRISPR-Cas systems." Nature biotechnology31.3 (2013): 233. PubMed PMID: 23360965...

... just hides in latently infected cells. To truly cure HIV-1, researchers need to vanquish these hidden...for HIV-1 patients. However, ART is not an HIV-1 cure. In patients who stop ART, viral levels will soon... the percentage of cells with activated HIV-1, measured via HIV-1 promoter-driven GFP expression. In cell...target sequence. Kaminski et al. next tested their procedure in HIV-1 infected T-cells to see if they could...therapies target HIV-1 at the level of protein structure, CRISPR gRNAs require DNA sequence-specific binding... note that these experiments were done in cell culture, and the cells infected with HIV-1 only after stable...papers present tantalizing evidence that an HIV-1 cure may be within our reach. Similar studies have shown...

...be boiled down to one, key concept: Creating a culture of excellent, effective communication between all...on the team talks and listens in roughly equal measure, keeping contribution short and sweet. One key... face one another, and their conversations and gestures are energetic.  This can take practice and training...training. This is important because it creates a culture of engagement in the issues at hand and eagerness...tactics for effective delegation will be covered in a future post in this series). Members carry on back-channel...talking about gossip here – this means encouraging a culture of maximum information flow so everyone knows what...

... you and the original depositor. Our citations feature makes it so that, when you cite a plasmid from ...to find your work later. We also hope that this feature will decrease redundancy. If you modify a plasmid...section. We’ve also added the Plasmid of the Day feature to our home page - each day we showcase a new plasmid...plasmid that you might otherwise miss. We hope this feature will draw attention to the great variety of plasmids...repository. And last, but certainly not least, we often feature new plasmid technologies on our blog and encourage...Evolving Check out the Blog Post on Our Citations Feature Browse More Inside Addgene Posts Additional Resources...

...of time, especially at room temperature or at 4°C. Storing DNA: temperature and longevity You should consider...the DNA. pH and DNases affect DNA stability Figure 1: So many choices! What to do? The main factors...are pH and DNase contamination.  Extreme pH can denature, break, and even change the DNA sequence. High...elution choices: TE, Tris buffer, or water Figure 2: Tris-EDTA, Tris, or water? The buffer you choose...Plasmid DNA can be stable at 4°C or even room temperature for a short period, and there are indications...

...not terribly problematic when working with cell culture, because it is easy to harvest large quantities...sheering of DNA by sonification. Though it helps capture weaker or more transient DNA-protein interactions...of interest. First, nuclei from tissue or cell culture are isolated using lectin-coated magnetic beads...thus targeting MNase to antibody bound proteins (Figure 1). Importantly, since Protein A has weaker affinity...desired. It also provides an excellent additional measure of DNA binding sites in combination with existing...fusion proteins Study chromatin interactions with CAPTURE Additional Resources on Addgene.org Find simple...

.... And make sure to subscribe to the Career Channel of the blog so you don’t miss the future posts in this...eScience Labs.  “We work with faculty members to make sure the courses encompass what they want to teach.” ...onsite, Caitlin can visit the operations team to make sure “that everything that’s going into the kit is going...manager also requires Caitlin to look at the big picture: “It’s been really cool to take a step back and...science. And I do it well.’” Stay tuned for the next feature in this series. We’ll be interviewing Susan Keown...

... comments. They could be featured in future career blog posts. And make sure to subscribe to the Career...Iowa, he was like a lot of newly minted PhD’s: not sure what his next step should be. He debated taking ...few other tips: “Learn HTML, write a lot to make sure you don’t hate it, and be flexible. The best stuff...appealing images and colors draw people in. Drawing a picture of a mechanism that also looks nice is a great ...

...mysterious source of failure the lab gnome. See this video where he/she/it/something is captured on camera: ...taking many pictures, one for each position in a movement, and assembling the many pictures together to... our free time. After taking almost a thousand pictures, my husband Enzo will spend a few days editing...you an idea of the amount of work, see how many pictures we take to make a walk cycle of one step. Challenges...

...hire and fire to make sure the team is the best it can be. Managers must ensure that their teams are working...presentation many times, I could never actually figure out what skills were really leader-specific. How...doer mode even late in our careers.  Managers make sure the work gets done Managers do this by removing ...about her management style. Leaders look to the future Leaders must get new things started and then step...the driving inspiration, to create a productive culture and to correct systemic problems. Understanding...

...cellular events such as the dynamics of subcellular structures and gene expression. You must see the movies ... a compatible fluorescent protein. FRET can be measured by exciting the most blue-shifted fluorescent ...optogenetic receptor, melanopsin, requiring brief exposure to 470nm blue light. To avoid overlap with this...gene tagging using a ligase-4-dependent mechanism. Nature Comm. 7, 12338. PubMed PMID: 27465542. PubMed Central...microscopy and enhances bioluminescence imaging in vivo.Nature Biotechnol. 34, 760-7. PubMed PMID: 27240196....luminescent protein for real-time multicolour bioimaging. Nature Comm. 7, 13718. PubMed PMID: 27966527. PubMed Central...NanoLuc-based luminescence (BRET) Ca++ sensing. Nature Comm. 7, 13268. PubMed PMID: 27786307. PubMed Central...

... foreseeable future and is a great way to get ready for the live audiences in your future.     3. Start...months, you may be looking for more ways to make sure you are moving forward. Here are a few out-of-the-box...yourself. You can watch yourself to critique and figure out what to improve. You can share it with others... The conversation builds a relationship for the future and the 1:1 format is good for everyone including... both building relationships and thinking about future career options. 5. Write a grant - even if you ...

...inherently unstable due to their secondary structure, palindromic nature, and high GC content. This instability...plasmid and take a look at each of its parts. Figure 1: Parts of an AAV transfer plasmid. Parts shown... sequence through ITRs due to their secondary structure and high GC content, but some companies now offer...spatial and temporal activation of a transgene. Figure 2: FLEX Cre-Lox system.  WPRE The Woodchuck ...Posttranscriptional Regulatory Element (WPRE) forms a tertiary structure when transcribed which aids in nuclear export ...

...viral-mediated. Transformations are usually bacterial in nature. All three of these methods involve DNA incorporation...be flanked by viral packaging elements that will ensure the virus gets produced, and viral particles are...functional viruses later on. These built-in safety features provide extra layers of risk management when working...floating in it, and then sterile filtered to be extra sure nothing untoward gets transduced (like a rogue HEK...virus, cell type, and/or desired event.   Figure 1: Transduction to expression process. A viral ... the right method for your experiment.  A cool feature of viral work is that you can swap out your virus...

...the extremely long, highly structured HCV genome (~9.6 kb of highly structured template!). MarathonRT also...localization in future studies of other genomic loci. Find the C-BERST plasmids here. Gao at al. Nature Methods...involved in transcriptional regulation and chromatin structure at your locus of choice. Find the Tet-inducible...Tet-inducible caspex plasmid here. Myers SA et al. Nature Methods. 2018. PMID: 29735997 bioRxiv preprint posted...

...career outside the traditional academic silo to tenured professor.  Non-academia encompasses millions of...he would give up,“an excellent—some say “cushy”—tenured faculty position for the grind of corporate life...industry but the “product” is publications—the pressure to produce is the same You get to interact with...business hours (mostly), but can often achieve more structured time management conducive to work/life scheduling...short and exciting ride Complicated matrix structures are common Small integrated teams, little hierarchy...interpersonal issues (one bad apple can change entire culture) Specific requirements for hiring more common...

...cleavage in mammalian cells, likely due to high temperature requirements. To solve this problem, Gao et al...NgAgo head to head NgAgo has a number of unique features compared to Cas9. These include: More targeting...vitro transcribed, and changes in gRNA secondary structure can affect editing efficiency. Gao et al. tested...1228 or 1307 aa depending on the species). This feature is advantageous if you’re working with small capacity...excision would alter the target sequence and preclude future HDR, which could lower HDR efficiency. Higher sensitivity...DNA-guided DNA interference by a prokaryotic Argonaute.” Nature. 507(7491) (2014):258-61. PubMed PMID: 24531762...