...applications. We recently released a new variant of jGCaMP7 (keep reading) and will be releasing more variants in...viral vectors. Here are some of the AAV we have released so far in 2019. You can also browse our entire...pAAV-mDlx-NLS-mRuby2 CAG-NLS-GFP pAAV-hSyn-mCherry Read on to learn more!  Calcium sensor AAV The GFP-based calcium...inhibitors that respond to blue light. We recently released a constitutive (pAAV-Syn-Archon1-KGC-GFP-ER2) ...coming soon! These vectors listed below should be released at Addgene in the next 6 months, pending quality... particular catalog item is available for order please email [email protected] with the vector name and...viral vectors 101 eBook Resources on Addgene.org Learn about Addgene's viral service Find viral protocols...

...This decrease in pH quenches SEP but not DsRed, leading to emission of only red fluorescence. The ratio...ratio of red to green fluorescence allows for clear distinction between vacuolar/lysosomal and cytoplasmic...lysosomes. Therefore, the formation of autophagosomes leads to an increase of yellow puncta (GFF positive/RFP...autophagosome with a lysosome. An increase in autophagy leads to more yellow and red puncta, while a block at ...at fusion of autophagosomes with lysosomes leads to an increase in yellow puncta but a decrease in red ...Addgene! GFP-LC3-RFP-LC3∆G: A fluorescent probe that releases an internal control Another autophagy biosensor... following translation, this fusion protein is cleaved to generate one molecule of GFP-tagged LC3 for ...

...classroom or a defined learning program. Learners cannot (theoretically, at least) fully and freely engage... fall under informal learning; however, many others partner with formal learning to engage students at...education that is provided, typically ad hoc, for learners who can choose to engage or disengage with a very..., and their expectations of you, consistent and clear.   And now that we’re all on the same page, we’re...re ready to get into the more exciting parts - learning more about science communication itself! See you...

... said Claudia OuYang, BeHEARD Co-Director in a release. The research teams studying four of those rare...disease caused by mutations in the OCRL1 gene that leads to the early death of young patients as their kidneys...processes involving cell membrane dynamics. When they learned there was a disease associated with the gene, it...Pigmentosum Hendrik De Buhr (top right) and his colleagues at the Wistar Institute in Philadelphia, PA will...pigmentosum (XP). This recessive genetic condition leaves patients incredibly sensitive to sunlight and prone... help them to understand how different mutations lead to differences in the symptoms experienced by AHC...AHC patients. Waardenburg Syndrome Last but not least, Meng-Shin Shiao (right) of Mahidol University’s...

...and knew it was time to leave. I really enjoyed my time in the lab, my colleagues and what I was researching...way to learn something about marketing would be to start reading a broad simple overview, learn some jargon...www.phdadvice.com. I sold out, well in part that’s what leaving academia feels like for a lot of researchers. For...years I struggled with the questions of “Will I leave academia?” and "What type of science careers would...Career Achievements”, and “Academic Achievements”. Leaning my CV more towards me as a product and not me as...important. For my Postdoc position, I tried to read at least 50 papers on the topic before I went for the interview...

...additional nuclear localization sequences; and two mutations in Cas9 previously shown to improve nuclease activity...Previously developed genome editing methods using nucleases and base editors have the potential to correct...precise changes. However, the efficiency of Cas9 cleavage is relatively high while the efficiency of HDR...inhibiting this complex may allow time for 5′ flap exonucleases and DNA ligases to act first. PE4 and PE5, as...editing in mammalian cells: the small RNA-binding exonuclease protection factor La. They found that La, which... ~ ~ ~ ~ ✓   ✓ SV40 and c-Myc nuclear localization sequences Improve translocation ... R221K N394K Cas9 mutations Improve Cas9 nuclease activity ~ ~ ~ ~ ✓ ✓ ✓ Evolved...

...switch that expresses nuclear mCherry in the absence of Cre, and expresses nuclear EGFP in the presence...viral vectors. Here are some of the AAV we have released in the last few months. You can also browse our...testing purposes. To address this, we recently released our serotype testing AAV, which are small (20 ...continue to add more controls, and have recently released these controls: pAAV-hSyn-DIO-EGFP (50457-AAV5...and recombinase-dependent vectors. We recently released several recombinases in various serotypes, and...grow this collection. Of note, we are excited to release a Flp-dependent Cre AAV in AAV9 and AAVrg. See ...viral vectors 101 eBook Resources on Addgene.org Learn about Addgene's viral service Find viral protocols...

... in order to protect their DNA from host cell nucleases (4). To further complicate matters, even when ..., and Szymanski in January 2016 in Viruses (5). Please refer to the paper for more details on the assay...short recognition sequence (like MluCI) that will cleave DNA frequently enough to result in a smear of fragments...inserts. We consider a library worth using if at least 40% of colonies tested showed correctly sized inserts...membranes will then be exposed to a lysis buffer to release recombinant proteins directly onto the nitrocellulose...solution-soaked filter paper (colony-side up) and leave for 1 h at room temperature. After this, it is helpful...min at 37°C (each repeated 5 times) works well. Leaving the membrane overnight in the freezer during one...

...Previous Post on CRISPR Mouse Models Learn about Homology Directed Repair Learn about Cas9 Ribonucleoprotein ...previous methods yielding 1-10% knock-in. Read on to learn how you can make CRISPR mouse model generation easi-er...re fortunate that there’s a guide that directs cleavage right before the stop codon!  Figure 3:...arrows) and their PAM (bold highlighted text) and cleavage sites (slashes) are shown. In this case, the green... green guide would be selected, since it cleaves at the stop codon TAG. Design your repair template...Miura et al. (2018) recommend targeting sites at least 100 bp away from the target exon on either side....in a two-guide floxed experiment, if one guide cleaves less efficiently than the other, a partial insertion...

...reproducibility at Addgene and here I summarize what I learned both in preparation for my talk and at the minisymposium...science and research. Nonetheless, I’d like to steer clear of saying we have a reproducibility “crisis” on ...irreproducibility with some solutions based on what I’ve learned. I hope you can apply some of these solutions to...interactive protocols that make it much easier to learn new experimental techniques. Electronic lab notebooks...perfect story for a “high impact” publication can lead to the publication of rare flukes showing positive...begin requiring reproducibility checklists that at least make it obvious that reproducibility is a priority...performing experiments with too few replicates leading to suspect statistical significance (you can read...

... not be too leaky. For inducible prokaryotic promoters, pLac is known to be slightly leaky, and pBad is..., and light are all examples of factors that can lead to the induction of a promoter. Below, you’ll find... cAMP and represses pBad, decreasing promoter leakiness.   Other examples of chemically induced promoters...sensitive expression systems are typically less leaky than chemically induced promoters; they show near-zero...heat shock. In the case of Hsp70, the heat shock releases heat shock factor 1 (HSF-1), which subsequently... be compatible with your experimental system. Leakiness If you’re expressing a gene that may be toxic,...inducible promoters are also known for their low leakiness. Inducibility level How much do you want to induce...

...Cell Stem Cell, Musunuru, Chad Cowan and their colleagues describe a much more efficient tool for doing...cells: a transcription activator-like effector nuclease (TALEN) kit consisting of 834 plasmids. The researchers...proteins that comprise a DNA binding domain and an endonuclease domain. What’s distinctive is that the DNA-binding... rest of the protein in place including FokI endonuclease domain as well as fluorescent proteins used ...first really popular tool was the zinc finger nucleases. Unfortunately, it is time consuming and difficult...without a lot of expertise to create zinc finger nucleases that will bind desired sequences. TALENs are much... genome. It remains to be seen which system is cleaner in terms of minimizing off-target effects, but ...

...first experience, I learned the details of grant writing, volunteered to take the lead on various applications... Thus, every single sentence and figure must be clear, concise, meaningful, and supportive of the mission...funded proposal will address these limitations and lead to new milestones in the field. Research strategy...that the goal and the impact of the project are clearly stated and align with the description of the RFA...see what tips they found useful. Step 4: Read and learn from the reviewers Reviewers may take 6-12 months...application. Although some comments may make it clear that the reviewer misunderstood the point that was... being made, take it as an opportunity to make clearer statements on the next round of applications. ...

...conditions, the protease cleaves itself and the degron off at the protease cleavage sequence. The tag is ...function, the SMASh tag self-cleaves itself when you want the protein around, leaving a minimal additional sequence...asunaprevir, the viral protease can no longer self-cleave and the protease and degron remain attached to ...protein-of-interest and the CRBN E3 ligase complex leading to ubiquitination and proteasomal degradation. ...HaloTag is quite large (33 kDa) and does not self-cleave off of the protein like the SMASh tag does. However...round of evolution (AID-2) to reduce some of the leakiness associated with the system, but the general concept...

...stage with much-lauded CRISPR-Cas9 is DNA-guided endonuclease NgAgo! We'll discuss how NgAgo is faring with...regions of the genome, which seem resistant to Cas9 cleavage (1). Both the CRISPR-Cas9 and NgAgo genome engineering...mammalian cells and one which diminishes the endonuclease activity of the enzyme. In vivo, only nascent...be rapidly degraded by the cellular machinery, leaving a small amount of active NgAgo/ssDNA complex in...manages to form a functional complex with ssDNA leading to inefficient downstream editing. Overall, the...suggestions and tips with #NgAgo The Addgene Blog - Leave suggestions and tips in the comments section below...someone came up with a mutant NgAgo (or a novel endonuclease) protein that can form a complex with a 5’-P-ssDNA...

...will be to your colleagues. If you ever have any questions about our plasmid pages, please don't hesitate...best use of all the information provided by your colleagues. Top of the plasmid page The ‘breadcrumb’ trail... is listed on the plasmid page.  The paper link leads to a page listing all the plasmids from this paper...References sections when citing the use of your colleague’s plasmid. Use the Addgene catalog number to easily...which experimental systems it was validated in.   Please keep in mind that all of the information on our...Check out Our Walkthrough of the Deposit Process Learn How We Do Quality Control at Addgene Resources ...Resources on Addgene.org Join the Addgene Community Learn the Benefits of Depositing Browse the Repository   ...

...classification system for the array of CRISPR technologies. Learning the general principles of this classification ... Cas3. Cas3 functions as both a helicase and a nuclease, unwinding and cutting up DNA after recruitment...systems are also employed as CRISPR transposases, by leaving out Cas3. Type I is further subdivided into subtypes...and DNA, though it is largely believed that the cleavage of DNA is the primary function of this type of...consider to be an integral component of CRISPR — a nuclease effector. However, IV-C does in fact have a helicase...caused by Cas12 create short 3’ overhangs, which leads to slightly increased efficiency when conducting...and binds the RNA target before initiating RNA cleavage. Created with BioRender.com.   What’s your...

...for Science Learn How Future of Research is Advocating for Change Within the Sciences Learn How Changing...partisanship, including more scientists in our leadership, and raising awareness regarding the pitfalls... Nicholas Thompson is a PhD Candidate studying Nuclear Science and Engineering at Rensselaer Polytechnic...women of reproductive age live in a state with at least two abortion restrictions that conflict with scientific...local law enforcement, and, although there was at least one person vocally protesting us, he was not threatening...I marched to advocate for the process we use to learn about the world. In a way, I marched for truth. ...that I loved - there were booths for children to learn little things about science. We need to keep encouraging...

...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. OpenWetWare Wikis Website: ... using. For the novice, the format does have a learning curve, but OWW walks you through the process with...

...addition to this cis cleavage, the authors showed that Cas14 could indiscriminately cleave ssDNA in trans like...variants share the presence of a predicted RuvC nuclease domain, characteristic of CRISPR-Cas enzymes. ...and more complex Cas9 and Cas12 proteins. Cas14 cleaves ssDNA But what is the function of this new system...bacteria strain. They found that Cas14 could bind and cleave a targeted sequence of ssDNA. Unlike Cas9, Cas14...for instance, the enzymes are activated and can cleave fluorescent probes of dsDNA and RNA in trans. Fluorescent...30337455. Additional resources on the Addgene Blog Learn more about DETECTR Browse all of the CRISPR blog...