...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...

... oligo: 5’- CACCGNNNNNNNNNNNNNNNNNNNGTTTGGGTCTTCGAGAAGACCTATTCXXXXXXXXC -3’; Reverse oligo: 5’- AATTGXXXXXXXXGAATAGGTCTTCTCGAAGACCCAAACNNNNNNNNNNNNNNNNNNNC...Cell 163.6 (2015): 1515-1526. PubMed PMID: 26627737. 5. Koike-Yusa, Hiroko, et al. "Genome-wide recessive...How Barcodes Can Be Used in Lineage Tracing Read about CRISPR/Optogentics Tools Resources on Addgene.org...

...that adding an additional guanine nucleotide to the 5’ of 20-bp guide can increase targeting efficiency....that adding an additional guanine nucleotide to the 5’ of 21-bp guide can increase targeting efficiency....Addgene Zhang Lab CRISPR Resources Tips for and FAQs about the Zhang Lab CRISPRs Browse All Zhang Lab Plasmids...

...generates double strange breaks at target sites 5' of the 5'NGG3' PAM sequence as directed by a gRNA. 2A-...the Addgene Blog Browse Our CRISPR Posts Learn about Using CRISPR to Generate Knockouts Read Some Common...

...answer questions about the evolution of the genes involved in methionine synthesis (5). The budding yeast...PubMed Central PMCID PMC4596657. 5. Thomas, D., & Surdin-Kerjan, Y. Metabolism of sulfur amino acids in Saccharomyces...well-characterized pathway with multiple enzymatic steps (5). In their experiments, students test whether or not...Addgene Blog Learn about Synthetic Biology with BioBuilder Use Addgene Videos to Learn about Basic Lab Techniques...species, but these studies generally leave questions about gene function wide open. What if two seemingly similar...question, the plasmids are also used to teach students about restriction mapping and western blotting. Students...communicating science to the public. You can find out more about Clare at the Biology Dept. site.  References 1. ...

...are driven by narrative and story telling. Chapter 5: The SITN Blog - Vini and Amy describe how the SITN...Science Careers 10 Steps to a Perfect Science Talk 5 Reasons to Use Reddit for Science Communication ...To learn more about the STIN's podcast, email [email protected]    For more info about starting your...evens where scientists can grab a beer and chat about their work at a local bar. It doesn't have to be...1: Introduction - Vini and Amy tell us a little about themselves. Chapter 2: What is Science in the News...science communicators. In this chapter we learn about some of their events - science seminars, science...the lab and working with SITN. She points to collaboration and delegating as keys achieving success. Chapter...

...This is the second in a 5 part series on Management for scientists. This second installment in the Management...feedback pretty quickly.   This is the second in a 5 part series on Introduction to Managing People for...post, a study coming out of MITs Human Dynamics Laboratory identified five characteristics of very successful...conversations within the team. We are not talking about gossip here – this means encouraging a culture of...Medical School researcher Connie Cepko to learn about her management style.   Seeking feedback frequently...information to make change whenever possible. Be public about changes influenced by feedback. If you don't do ...

...plasmids pX330S-2 to pX330S-(last gRNA number). The 5’ most gRNA is cloned into the Cas9-containing destination...Multiplex Plasmids: These plasmids allow expression of 2-5 gRNAs in zebrafish. Custom destination vectors are... CRMAGE is an incredibly fast protocol, with only 5 hours incubation time needed for a single round of...PMC4396471. Find plasmids from this paper at Addgene. 5. Tsai, Shengdar Q., et al. “Dimeric CRISPR RNA-guided...National Academy of Sciences USA 112(11) (2015): 3570-5. PubMed PMID: 25733849. PubMed Central PMCID: PMC4371917...multiplexing options, but also check out our blog post about multiplexing with Cpf1.   Figure 1: Multiplexing...assembly.  Unlike some of the plasmids described above, this vector does not contain Cas9, so you’ll need...

... fourth plasmid. In addition, the promoter of the 5’ long terminal repeat (LTR) is mutated in third generation... size. Opting for a larger vessel, such as a 3 or 5-layer flask will require some initial optimization...preparations without ultracentrifugation. Sci Rep. 2015; 5:13875. PubMed PMID: 26348152. PubMed Central PMCID...available systems. Producing lentivirus: it's all about the T's (transfection and titers) To produce lentivirus... optimize transfection conditions. As mentioned above, plasmid ratios can be adjusted to enhance titer...

... gene promoter and the MYC locus (Figure 5). Figure 5: TurboCas protein labeling compared to immunoprecipitation...insertions up to 12 kb — with efficiencies between 5% and 11%. It has higher efficiency and lower indel...peptide (VIP), or lysosomal-associated membrane protein 5 (LAMP5) — as well as cholinergic (choline acetyltransferase...insert your genetic cargo of choice. After hearing about researchers' favorite features and limitations of...development and cancer. But there is still much to learn about their myriad functions and therapeutic potential...created a very quick and efficient way to learn more about gene regulation. Find the TurboCas plasmid here!...Initiative Armamentarium Collection, a major collaborative effort to develop molecular tools for studying...

...terminators and promoters with buffer sequences on their 5’ and 3’ ends (for insulation). A list of promoters...interference between the transcribed gene and its 5’ UTR. Various self-cleaving hammerhead ribozyme parts...recombinases have been described in the literature (5, 6) and may be substituted for one another in an RSM...PMID: 23034349. PubMed Central PMCID: PMC3914141. 5. Brown, William RA, et al. "Serine recombinases as...certain cellular behaviors, but little is known about the order or timing of these factors. Recombinase-based... three methods of state interrogation mentioned above (Sanger sequencing, qPCR, and fluorescent reporter-based...Bac Vectors Can Be Used for Genome Editing Learn about Michael Koeris' Journey from Grad Student to SynBio...