We narrowed to 1,015 results for: LEA

...Cre-tagged mitochondria, Cre is released, translocates to the nuclease, and excises the DsRed-STOP to ...neurons interact, the labs of Baljit Khakh and colleagues mapped their cell-surface proteomes (Wu et al...express iCre with a mitochondrial anchor and a TEV cleavage site. Recipient cells express DsRed within a lox-STOP-lox...Figure 2: Schematic of the MitoTRACER system. NLS = nuclear localization signal; TEVp = TEV protease. Created...These new sensors use quite different designs, leading to unique advantages. While most red GECIs have...extensive validation of the cell and gene therapy, so please check it out! Find AAV-CRISPRa plasmids here! ...translation of chromoprotein and gRNA plasmids. Nuclease-dead Cas9 (dCas9) protein is supplied separately...

... about roles in this field, most jobs needed at least 3 months experience working in a developing tropical...positive experience. I enjoyed helping people and learning to explain concepts in ways that people from another...traditional medicine couldn’t fight effectively. I had to learn how to cope with a different climate and culture...work became monotonous very quickly; as soon as I learned how to do the different tests and became efficient...where one drug does not work for all patients, leading to very high mortality. I really loved the fact...cannot be as adaptive. I was also surprised to learn that I enjoy the customer service side of my job...vital because otherwise I’d lose touch with my colleagues and have misunderstandings.  Working long distance...

...bound lipoproteins Article contributed by Leah Schwiesow A leading cause of death worldwide, cardiovascular...ever like to write about a recent plasmid deposit please sign up here.        A retroviral mouse CRISPR...genetically. To solve this issue, Sarah Teichmann and colleagues created a retroviral CRISPR knockout library ...lipoproteins through the circulatory systems, but can also lead to the formation of atherosclerotic plaques. ApoB...through triglyceride/cholesterol does not provide a clear view of either of these metrics (Sniderman et al...-LP size and concentration, Steven Farber and colleagues at the Carnegie Institute for Science have developed...

...of a paper is the one that you need to spend the least time on—just put down a basic outline and refer ...figure them out. But, when they come to light, it’s clear that what the researchers thought was a problem ...role, and we take our responsibility to push for clear communication of science very seriously. That’s ...and transparency of methods sections, including clear information about reagents, and why other publishers...want to be? The part that is still missing is a clear alignment of incentives. Right now, researchers ...evidence of sharing for grant renewal—but there is clearly room to do more. If we could really instill a culture...Her current responsibilities encompass overall leadership of the Cell Press editorial department, a team...

...miscommunication, a lack of leadership, and inappropriate tools or writing approaches can lead to frustration, delay...Weill Cornell Medicine. Discussing data with colleagues can be more difficult during the pandemic era...can contribute and sketch on the board without leaving Zoom. The annotate function can also make the screen-sharing...cultural backgrounds.” This work outlines the need for clear communication, fair credit through authorship, and...ways of working, reconnect as human beings and colleagues, eventually encouraging purposeful and creative...Medicine in NYC and author of the book “The Salmon Leap for PhDs: Swimming upstream a career from academia...

...describe a process that begins with cell lysis to release AAV particles with subsequent purification steps...also possible for AAV vectors to be spontaneously released from the producer cells into the supernatant during... are exosome AAVs? In 2012, Casey Maguire and colleagues from Massachusetts General Hospital and Harvard...took a closer look at the AAV vectors that are released from cells during production (Maguire et al., ...the exo-AAV isolation protocol by Maguire and colleagues, other researchers have tried to modify it to...possibly improve transduction properties. György and colleagues found that the association of AAV particles with...viral vectors 101 eBook Resources on Addgene.org Learn about Addgene's viral service Find viral protocols...

...often a hard question to answer if you plan on leaving academia, but don’t quite know what you do want...goal, and when you realise that your goal might not lead to a career you actually want, you can feel lost...Journal Editor Science Marketer Software Developer Learn Science Communication Skill from a Student Group...in the media and in films, which can often be misleading. Key aspects to determine are working hours (... the job require public speaking? Will you be leading a group or working as part of team? Will you be...application. Additional Resources on the Addgene Blog Learn How to Experiment with New Careers while in Grad...Koeris' Journey from Grad Student to Entrepreneur Learn to Network and Make the Contacts for Your Dream ...

...through October 19. The BioBuilder Learning Lab The BioBuilder Learning Lab is a fully equipped, state-of-the-art...and teachers in the U.S. and beyond. “I want to learn more about Synthetic Biology. Can you tell me where...capitalize on the excitement found through experiential learning. “Students may forget the technical details they... BioBuilder experience With its intentionally malleable curriculum, BioBuilder takes cutting-edge research...a project-based curriculum, BioBuilder expands learning outcomes by emphasizing the importance of critical...production of fire-resistant housing insulation, and cleaning up salt runoff from winter icing.BioBuilderClub...

... we've heard over and over from our users: "Please, please improve your plasmid maps!" After thoughtful... here to see the new map in action). Read on to learn more about the improvements we’ve implemented and...extraneous ORFs that cluttered our old plasmid maps. Clearly Annotated Features, Enzymes, and Primers - The ...easier for you to share plasmid information with colleagues, or explain plasmid features to a lab member ...Once you’ve identified a plasmid that you want to learn more about, updates to our sequence analyzer will...hover over any feature, enzyme, primer, or ORF to learn more information about it. For example, if you hover...

... Sample6 has changed focus and names, swapped leadership, and grown to 25 members.  What is Sample6?...competing in business plan competitions. After much learning, the focus of the application was changed, but...best, wins. He explained that entrepreneurs must clearly communicate their idea at various levels of complexity...industry vary drastically, he reassures that one can learn it by reading a broad variety of materials and constantly...million. The biggest challenge during this time was learning to communicate effectively to non-scientific audiences...are essential to the biotech startup puzzle: Nucleation of the team. The team is key, and the initial...raise money and/or validate an idea or product? Please let us know in the comments section! And read on...

...often terminate with a poly(A). ORF2 encodes endonuclease and reverse transcriptase activities.    ...site for integration is TA dinucleotides and it leaves behind the CAG DNA footprint from its terminal ...terminal sequences at the excision site after cleavage by the transposase. It has a cargo capacity >100 kB, although... TTAA and unlike other transposons, it does not leave behind a DNA footprint sequence after excision. ...   Applications of transposons Now that you’ve learned about some of the popular transposon systems, let...DNA into the pronuclei of fertilized eggs, which leads to random incorporation of that sequence into the...ijms22105084 Additional resources on the Addgene blog Learn about the INTEGRATE system for bacterial genome ...

...the Addgene Blog Learn more about FRET Tips for Using FRET in Your Experiments Learn more about Fluorescent...brightest since it had an emission near 600 nm – a clear demonstration of why a BRET reporter can improve...Many intracellular sensors of events like calcium release have been made using fluorescent resonance energy... molecules. FRET sensors face challenges of photobleaching, autofluorescence, and, in the case of exciting...wavelengths, excitation of the optogenetic regulator leads to spurious activation of the FRET sensor. Researchers...et al. (7) wished to trigger intracellular Ca++ release with an optogenetic receptor, melanopsin, requiring...#83926). Live-Cell Voltage Sensor  Ingaki and colleagues (8) were interested in using photoactivatable...

...nature of non-homologous end joining (NHEJ) often leads to the generation of indels and thus frameshifts...activation) and CRISPRi (inhibition) technologies – a nuclease-dead Cas9 (dCas9) is directed near the promoter...landscape of mismatches that can nevertheless still lead to activity has been established, and can be used...that the consequences of off-target activity, at least for the experimental conditions used, led to no ...the generation of truly isogenic cell lines than nuclease-mediated off-target effects.” Further, large-scale...al., 2019). Again, the experimental strategy is clear: for any gene of interest, one should require that... Liu DR (2020) Genome editing with CRISPR–Cas nucleases, base editors, transposases and prime editors....

...the RNases on your hands can degrade RNA. Keep a clean work area, which may include spraying your bench...Alternatively, glassware can be baked at 180°C for at least 4 hours.   If your final RNA sample(s) are resuspended...cell lysis.  In the late 1970’s, Chirgwin and colleagues showed that a strong protein denaturant, guanidinium...mindful of your extractions- if you need really clean RNA, it’s important to make sure that when extracting...effect of ammonium sulfate on the activity of ribonuclease A. Biochimica et Biophysica Acta (BBA) - Enzymology...active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18:5294–5299. https://doi.org/... Leah Schwiesow...

...macroautophagy of the cytosol leads to accumulation of Pho8Δ60 in the vacuole, where it is cleaved to generate an active... molecular weight shift of Pho8 from its uncleaved to cleaved form is the final read out of this assay...cytosol, to the higher pH of the vacuole. Read on to learn more about prior methods for studying autophagy ...the vacuole via the secretory pathway, and then cleaved to generate an active form of the protein. To monitor...lysosome, since both Atg8p and LC3 are degraded or released once an autophagosome fuses with the vacuole or...

...requisite for a functioning lab, but it is for a scaleable lab. When you need to track the location, quality...post we’ll highlight some of the lessons we’ve learned over the years with the hope that our insights ...containers and contents Right from the get-go be as clear as you can be (there will always be confusion about..., tightly coupling them to their contents might leave you sad and confused when a tricky procedure failed...worm that requires a change of tube, there’s no clean way to separate the contents from the container ...etc.There are also going to be situations where you learn biology terms (plasmid, 5-prime, CRISPR, growth ...krisshamloo.com     Additional Resources on the Addgene Blog Learn about How We Use Barcodes in the Lab Get Tips on...

...the Addgene Blog Learn How Scientists Are Trying to Shape the Future of Research Learn How Notions of Scientific...studies with concrete examples of how gender analysis leads to discovery and innovation. One such case study... of their cells when doing experiments—which can lead to failed research. For example, an international...develop represent the whole human population. To learn a little more about where differences in effects...There is much work to be done! Researchers need to learn sophisticated methods of sex and gender analysis...Blogger Londa Schiebinger. Londa Schiebinger is a leading international expert on gender in science and technology...

...Ask openly for clear information on costs. Can you suggest some coaches? Yes I can. Please note that Addgene...to change jobs? Grow your professional presence? Learn a key new job skill? Finish your thesis? Knowing...that will help you. A good coach usually will be a clear communicator and make direct suggestions for improvement...She is also a very popular speaker and workshop leader. Lauren Celano of Propel Careers is not a professional...engagements and has many areas of specialty from leadership development to mentoring for women and under-...Guide Your PhD with An Individual Development Plan Learn a Little about The Academic Labor Environment from...

...mEosFP exposure to UV light causes an irreversible cleavage near the chromophore, resulting in emission of...to blue light also inactivates the chromophore, leading to a non-fluorescent state. Dronpa can be reversibly...quickly inactivated and less photons are emitted leading to dimmer fluorescence. In fact, many of the early...are being emitted, making it easier to capture a clear image above background. The majority of PA-FPs come...disrupting cellular function and localization, or leading to protein aggregation. Another important parameter...light used to activate the first PA-FP does not photobleach the second, and vice versa. Finally, as every...contrast, PA-FPs offer a reduced potential for photobleaching and phototoxicity that standard FPs might induce...

... 2019). Inhibiting DNA cleavage Example: AcrIIC1 binds to the HNH endonuclease domain of Cas9 and prevents...prevents target DNA cleavage (Harrington et al., 2017). Figure 1: Anti-CRISPR proteins (Acr's) can...loading, inhibiting DNA binding, or inhibiting DNA cleavage.   Five ways to anti-CRISPR proteins in your...timing experiments, the Doudna lab found that at least 50% of on-target Cas9 edits happens within the first...effects and cytotoxicity that result from excessive nuclease activity in tissues (Li et al., 2018). 4. Selection...Blog First time CRISPR user? Check out this post. Learn how to Choose the Best Cas9 Variant for Your Next...

...implanted into a female animal for gestation. Nucleases that induce double-stranded DNA breaks, such as...cells, which are then injected into a blastocyst. Nucleases can also be used to engineer knockout mice. ZFN...non-homologous repair at the site of DNA damage. These nucleases are microinjected into a zygote, which is then...introduce point mutations. While using targeted nucleases to generate an engineered mouse model has advantages...targeting vector alone, some challenges still exist. Nucleases may cause off-target effects on regions of the... random process, zygotes edited using the same nuclease may not result in genetically identical mice. ...about Cre-lox Read our Genome Engineering Guide Learn more in our CRIPSR guide ...

...anymore. Say hello to PAM-free and PAM-flexible nucleases! In this blog, we will review Cas enzymes, which... uses a PAM with no sequence constraints, or at least has looser or different requirements. Luckily, there...Nonetheless, in an AT-rich region, these Cas9s may still leave you choosing between efficiency and location.   ...display higher off-target effects than SpCas9, a pleasant surprise since the PAM flexibility could in theory...activity of SpRY was validated in double stranded cleavage and base editing applications, with high editing... NG Cas9 NG - SpG Cas9  NG  - SpRY nuclease  C to T Base Editor SpRY A to G Base Editor SpRY...  Nishimasu, H., et al.,Engineered CRISPR-Cas9 nuclease with expanded targeting space. Science 361,1259...

...including mitosis, mitochondrial fission/fusion, nuclear import, and neuronal trafficking. Although FPs ...Overexpression of fluorescently tagged proteins can lead to improper protein localization, protein aggregation...power of this system lies in the ability of the endonuclease Cas9 to create a DNA double stranded break (...the tag, selectable marker, and connector to be released (Fig. 1, Step 2-3). Since donor plasmids of the...directed repair after a CRISPR/Cas9 induced break. Learn more about these constructs and the cell lines they...Cas systems used for endogenous protein tagging, please visit our website CRISPR/Cas Plasmids - Protein...PMC4889673. Additional Resources on the Addgene Blog Learn about Fluorescent Protein Timers Check out Photoactivatable...

...DNA could lead to transgene expression in recombinase-negative animals, producing misleading results.  ...the plasmid used to create the viral prep. In a clean viral prep, like AAV-44362 shown below, more than...from these known impurities, as was the case for clean sample AAV-44362. In addition to these expected ...number of hits to the same sequence (gene). In a clean sample, there are typically fewer than 10 hits to...receive the best AAV vectors for your research. Please use the comments section below to let us know if...Topic Page Subscribe to Viral Vectors Blog Posts Learn How We Use NGS for Plasmid Quality Control Resources...

...that delivering RNA-guided nucleases or TALENs together with AdV donor DNA leads to a vast majority of AdV-modified...chromosomal region. The CRISPR/Cas9-derived RNA-guided nuclease-induced gene disruption frequencies in the various... and demonstrated by Manuel Gonçalves and his colleagues at Leiden University Medical Center are now available...DNA for accurate genome editing with engineered nucleases. Nat Methods 11:1051–1057 . https://doi.org/10.1038...Adenoviral vector delivery of RNA-guided CRISPR/Cas9 nuclease complexes induces targeted mutagenesis in a diverse...delivery of CRISPR/Cas9. Journal of Controlled Release 298:128–141 . https://doi.org/10.1016/j.jconrel...

...This DNA degradation portion can be bypassed by leaving out Cas3 — a tactic some CRISPR transposase systems...chop DNA. Cas3, however, has two jobs, one as a nuclease and one as a helicase. Following recruitment by...initial steps, Cas3 can perform its helicase and nuclease functions simultaneously, by continuously unraveling...unraveling dsDNA, pulling through the ssDNA, then cleaving the ssDNA strands. In this way, Cas3 eats away... into think it’s more powerful than it is. The nuclease domain of Cas3 is considered weak, especially ...However, by continually passing the ssDNA past the nuclease domain, Cas3 is able to repeatedly cut the DNA...thymine-rich regions on the non-target strand. Occasional cleaving of the target strand is not as well understood...

... PITCh plasmid to release the cassette, and an insertion-locus-specific gRNA cleaves genomic DNA. These...it’s become clear that the power of this editing platform lies in the diversity of nucleases and their .... For more mechanistic details on HDR and NHEJ, please see the linked blog posts. Compared to its counterparts...compare to CRISPR sticky-end insertion using the nuclease Cpf1. Since Cpf1 cuts in a staggered pattern, ...Experiment Additional Resources on Addgene.org Learn All about CRISPR on Our CRISPR Guide Pages Find ...

...Recently I learned that Addgene’s pLKO.1 cloning protocol is viewed around 3,000 times each month. I ...this plasmid, and if it still being used. After learning of it's continued popularity, I decided this protocol...thousands of other scientists using pLKO.1 variants learn about those updates? This is a problem many scientists... found in our Plasmids 101 blog series. Want to learn more about sharing your plasmids via Addgene? Check...lab for non-bench work – but is there a sterile, clean way to bring your phone into the lab? Maybe having...protocols to the site and will keep you updated as I learn to use the resource. Nature Protocol Exchange Website...I missed, let me know in the comments below and leave your thoughts on how the app works for your lab’...

... Developed by David Liu and colleagues, FokI-dCas9 expresses Fok1 nuclease domain fused to catalytically... to this system by engineering cells that lack release factor one (RF-1; Bacterial strain EcAR7) and minimizing...control for your experiments. For more information, please see Addgene’s information page for the Rinehart...neuronal signaling. For more information on DREADDs, please see the Roth lab's DREADD users blog. New ... expression plasmid that was used by Chen and colleagues for genome editing inXenopus tropicalis. (Guo...information on using the Platinum Gate TALEN Kit, please see the Yamamoto lab’s protocol for TALEN construction...mGRASP A Nature 2012 article by Jinny Kim and colleagues describes their efforts to map the location and...

...editors. They adjust the side groups of DNA bases and leave the core ring structure intact, allowing them to...guide RNA to base pair with the DNA target strand, leaving the non-target strand unpaired and accessible on...it the “edited strand.” Uracil excision repair leads to the first base transversion editor The story ...improved the CGBE: DNA polymerase D2 (POLD2), exonuclease 1 (EXO1), and RNA binding motif protein X-linked...included in the CGBE. They also developed a machine learning model capable of predicting editing outcomes of...that were already accessible. It's not entirely clear why this is, but it might point to a more dynamic... screens, target-library analysis, and machine learning. Nature Biotechnology, 39(11), 1414–1425. https...