... cleaves in a staggered fashion, creating a 5 nucleotide 5’ overhang 18-23 bases away from the PAM. Cpf1...some of Cas9’s shortcomings. Cpf1’s preferred PAM is 5’-TTN, differing from that of Cas9 (3’-NGG) in both...evolutionary classification of CRISPR-Cas systems. 5. Nat Rev Microbiol. 2015 Sep 28. doi: 10.1038/nrmicro3569...a cell culture model. What we still don't know about Cpf1 In their original paper characterizing Cpf1...Addgene Blog Catch Up On Your Cpf1 Background Read About CRISPR Competitor NgAgo Learn How You Can Use piggyBac...

...in Malawi 4:36-5:46 : How Kingdom ended up working in the life sciences and agriculture 5:47-9:35 : Kingdom's...more about Dr. Kwapata’s current work and thoughts on the need for more scientific collaboration across... connecting us with him. Learn more about our recent collaboration with Seeding Labs here. Special thanks...received an award from nonprofit and Addgene collaborator, Seeding Labs, for lab equipment that will help...Addgene Podcast.     Additional Resources Learn about Seeding Lab's Work with Lilongwe Univeristy of Agriculture...How You Can Contribute to the Addgene Blog Learn about Addgene Plasmid Donations for Rare Disease Research...

..., and a room of 5-30 faculty members. The session lasts up to an hour. You will get 5-10 minutes to present... should be mechanistic, doable by 1-3 people in 1-5 years, and contribute to resolving the overarching...solve the problem. What will you have accomplished in 5-10 years? You need to be able to describe what success...a single grant. Either way, think realistically about the time and resources required to feasibly accomplish...interrupted frequently by committee members. As mentioned above, plan to give your 10 minute overview and address... list of common questions, check out this eBook about applying for faculty positions. You need to practice...program. You should be proud of it and be excited about getting the opportunity to realize your vision. ...

...was provided by Addgene on 5/26/20 and contains requests from 5/22/14 to 5/19/20). Each paper has between...break, and COVID-19 on plasmid requests Figure 5: Inferred effects. Values are the inferred percentage...in his laboratory. Learn more here. Additional resources on the Addgene blog Learn more about plasmid...

...PHP.B. Gene Therapy, 25(5), 392–400. https://doi.org/10.1038/s41434-018-0028-5 de Backer, M. W. A., Brans...We found that, in this case, AAV1 (23%) and AAV6 (<5%, data not shown) were not good options while AAV8...Initiative, F. B. (2019). Setting your data free. Nature, 5–7. O’Connor, D. M., Lutomski, C., Jarrold, M. F., ...asked why they don’t share, many express concern about sharing negative data they cannot explain, perhaps...

...alternating between 5 min at -20°C (or -80°C) followed by 5 min at 37°C (each repeated 5 times) works well...without the need for genome sequencing or annotation (5). In the event that genome sequencing can be done,...Sacher, and Szymanski in January 2016 in Viruses (5). Please refer to the paper for more details on the...our planet, but we still know collectively little about how they infect and influence the evolution of their...

...09 - 5:58 : Specific Examples where Niroshi and Ben helped build their grad student communities 5:59 -...How did you hear about post grad opportunities and when did you start thinking about them? 18:30 - 19:... top if you leverage your community, think hard about picking a good mentor, and begin considering career...19:54 : How to think about picking a post-doc project 19:55 - 22:40 : Improvements that can be made to ...

...take to fuel more than 30,000 neuroscientists over a 5 day conference? I feel like here at the annual Society...of coffee would be consumed throughout the entire 5 day event. Though, to be fair, I think this is a conservative...learn about all the amazing research being done with plasmids. I heard an impressive talk about the use...pulls off such an inspiring, thought-provoking, collaborative event – all while keeping the party going! At...scientists we've spoken with are excited and passionate about making such tools open-access. Though it is time...

...derived from the P and V proteins of Simian virus 5 (SV5) (Randall et al., 1987). The epitope (GKPIPNPLLGLDST...characterization of monoclonal antibodies to simian virus 5 and their use in revealing antigenic differences between...assemble and catalyze the luminescence reaction (Figure 5). The researchers used the reporter to visualize biologically...cells in several different contexts.   Figure 5: Left: Mechanism of the PILATeS tension sensor. The...more feasible, the lab of Alessio Ciulli and collaborators developed CRBNmidi (Kroupova et al., 2024). ...stepwise, achieving mutation efficiencies of up to about 9% of colonies (102 to 104 times higher than traditional...Bentley Time to get flexible! Alex Dunn’s lab and collaborators have deposited a new mechanosensor called PILATeS...

...rmmAkm6PQd — Elizabeth Kenkel (@ElizabethKenkel) December 5, 2018 How social media has helped me in science...have tips for using social media as a scientist. 1. "5 Ways Scientists SHOULD Be Using Twitter" podcast from...include in each post I write for the Addgene blog. 5. "Social Media in Your Working Day" video from Science... advantage. It wasn’t until I wrote a blog post about a research paper that explored how scientists connect...saw a request for an Addgene guest blogger post about colony PCR, I signed up immediately. I had used ...science communication skills, while also learning about new scientific developments and helping scientists... found this paper. I wrote an Addgene blog post about it and shared the paper with my lab since one of...

...Jude JG, Zuber J, Gerlich DW Cell. 2017 Aug 24;170(5):956-972.e23 Screen identifies bromodomain protein...Jude JG, Zuber J, Gerlich DW Cell. 2017 Aug 24;170(5):956-972.e23 Rapid and phosphoinositol-dependent binding...Swiderek K, Kuo A, Crabtree GR Cell. 1998 Nov 25. 95(5):625-36. An architectural role for a nuclear noncoding...Leah Bury of Microscopic Art. I started thinking about this problem when I recently attended the 2018 Chromosome...lot of scientists who not only remember to think about DNA in this context, but also specifically study...plasmids containing these genes. As with the PIs above, many of these genes were discussed at the 2018 ...chromatin This is not the first Addgene blog post about chromatin--multiple Addgene colleagues and guest...

...products are rare, probably accounting for less than 5% of repair events. However, the products of accurate...Break Diminishes Its Mutagenic Potential. PLoS Genet 5:e1000683 . https://doi.org/10.1371/journal.pgen.1000683...resolved during nonhomologous end joining. Nat Commun 5: . https://doi.org/10.1038/ncomms5286 Yan M-Y, Li ... extremely heterogeneous. There is consequently about a two-thirds chance of causing a frameshift mutation...favor accumulation of the latter products. As noted above, a single cycle of cleavage and accurate repair ...

...market; you need one with 5’->3’ polymerase activity (for amplification), 3’->5’ exonuclease activity (increases...(increases amplification fidelity), and no 5’->3’ exonuclease activity (which could potentially truncate...unique site in the coding region (preferably near the 5’-end) and that also generates 4 bp overhangs. You ... Brush up on Your Cloning Techniques Learn More about Plasmids in Our Plasmids 101 Series Use CRISPR to...

... yes   URA3 pyrimidine (uracil) yes - Grow with 5-FOA. S. cerevisiae yes - This can complement ura4-...required. yes   TRP1 L-tryptophan yes - Grow with 5-FAA. S. cerevisiae no  no TRP1 alters some yeast phenotypes...media. ura4+ pyrimidine (uracil) yes - Grow with 5-FOA*. S. pombe  no no *FOA in fission yeast induces...to high copy numbers. This creates a potential metabolic burden on the yeast cells. Some phenotypes may...

... positions available in academia (5, 6). Academic data and the labor market for PhDs Later this week, ...postdocs had achieved tenure-track positions within 5-7 years of earning their PhDs (3), showing that the...PMID: 25925677. Pubmed Central PMCID: PMC4484856. 5. Bourne, Henry R. "A fair deal for PhD students and... the Jackson Laboratory for Genomic Medicine and a Senior Research Associate at the Labor and Worklife...increasing data collection about the scientific workforce, in particular about postdocs. Among other involvements...Research Symposium Boston 2015 – Academic Data and the Labor Market. For more information on the symposium as...generating too many trainees per PI” “Lack of awareness about how the system operates and functions” “Complete...

... single fluorescent biosensor. Scientific Reports,5(1). doi:10.1038/srep12397. PubMed PMID: 26215030. ...206. doi:10.4161/auto.3678. PubMed PMID:17224625. 5. Noda, T., & Klionsky, D. J. (2008). Chapter 3 The...Systems, Part A, 33-42. doi:10.1016/s0076-6879(08)03203-5. PubMed PMID: 19185711. Additional Resources on the...information about the contents of the autophagosome nor does it provide information about what is being...higher pH of the vacuole. Read on to learn more about prior methods for studying autophagy and how Rosella... (as opposed to giving more fine-grained detail about what proteins or organelles are being degraded) ...autophagy, but, like the protein degradation assay above, it’s slow and only measures bulk autophagy. Morphological...

... known as sticky ends. Cas12a cleavage results in 5 bp overhangs and is particularly convenient for targeting...Cells Using Type I CRISPR-Cas. Molecular Cell, 74(5): 936-950.e5 (2019). 10.1016/j.molcel.2019.03.014 ...Nature, 597, 720-725 (2021). 10.1038/s41586-021-03886-5 Additional resources on the Addgene Blog CRISPR... is smaller than the traditional SpCas9 enzyme (about a third of the size) and does not require a tracrRNA...Cas12a to further optimize it for therapeutic and laboratory usage. Fast facts – PAM requirement: TTN or ... the lack of collateral cleavage). You can read about Cas7-11 in our blog post on its development and ...

...Requires tracrRNA? Yes No dsDNA cleavage Blunt end 5' overhang PAM sequence NGG TTTV PAM site preserved...preserved? Usually Destroyed Yes, Cpf1 cleaves 5' of the protospacer Multiplexing CRISPR-Cas9 options ... an example of how this works. Make sure to order 5’ phosphorylated oligos or treat with T4 PNK. Delivery...targets compared to Cas9.  Read on to learn more about Cpf1 multiplexing.  For an in-depth review of Cpf1... Addgene. If you have any questions or thoughts about Cpf1 multiplexing, leave them in the comments below...

...interest and has an average off-target frequency of 5%. Your experiment involves making a large sgRNA ...1,000 targeted cells to sequence. In this instance, a 5% off-target frequency is something to keep in mind...used in human clinical trials. An off-target rate of 5% is far too high when considering human patients and...your off-target frequency to sit at a level of, say 5%, then the majority of single cell editing events ...is an off-target effect. Should I be concerned about off-target effects? Depending on your experimental...this could be a huge concern, or nothing to worry about all. Let’s consider several scenarios below for ...Predicting off-target events As mentioned in the above section, there are gRNA softwares available which...

...different enzymes: an exonuclease, which chews back the 5’ ends of the fragment, generating long overhangs which... get primers, you can have your construct made in 5 days. One drawback to the Gibson assembly technique...aspects of the SLIC methods. First, it uses a dedicated 5’ exonuclease instead of using the exonuclease feature...PMID: 23663447. PubMed Central PMCID: PMC3684516. 5. Casini, Arturo, et al. "One-pot DNA construction ...the annealed and filled-in gaps The great part about this mix of enzymes is that they can all work at...the standard Gibson assembly techniques described above, this resulted in the direct and seamless cloning...Blog Browse All of Our Plasmids 101 Posts Read About Other Cloning Techniques Additional Resources on...