... 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...

...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...

...SpCas9), potential target sites are both [5’-20nt-NGG] and [5’-CCN-20nt], as it is equally efficacious ...occur ~1 in every 8 nucleotides, restricting gRNAs to 5 – 65% of the protein coding region will still result... A>G base editors, the intended edit must be in a 5 - 10 nt window relative to the PAM, and bystander ...target should be near the TSS but you can worry less about sequence optimality because you generally have fewer...

...non-coding RNA genes. Nature biotechnology. 2020;38(5):573. doi:10.1038/s41587-020-0428-0 Rinn JL, Chang... sites. Nat Biotechnol. Published online November 5, 2018. doi:10.1038/nbt.4283 Zibitt MS, Hartford CCR...et al. Small indels induced by CRISPR/Cas9 in the 5’ region of microRNA lead to its depletion and Drosha... to devise an experiment that can tell you more about its function.  Resources and references Resources...

...Education. 38(5):324-329. Kowalski, J., Hoops, G., Johnson, J. 2016. Implementation of a collaborative series... diversify science. Evolution and Development. 24(5):127-130. Kimball, C., Powers, K., Dustin, J., Poirier... Biochemistry and Molecular Biology Education. 38(5):317-323. Knutson, K., Smith, J., Nichols, P., Wallert...in a year-long biochemistry laboratory : Part II – research-based laboratory – a semester-long research...are often associated with rote memorization of metabolic pathways (precisely the type of exercise Einstein...had the opportunity to talk with Provost recently about his development of what is now commonly termed a...into one of my own courses, I was excited to hear about how he developed his program. Initially, Provost...

... Be realistic about what you can teach  5-10 year olds in a single evening. Grades K-5 cover a broad ...school system that has made strong efforts in the last 5 years to incorporate more science and technology into...wonderful tangents. Some groups asked about vaccines, others were curious about the agricultural repercussions...roles and responsibilities, I have to think a lot about how to balance those roles. I’m a father of three...community. This past year, I spent some time thinking about how I could combine all of these roles by reaching...guest presentations. If you’re a scientist thinking about reaching out to your community’s schools, here are...DNA, protein, plasmid, gene, etc. Though I talked about CRISPR’s molecular mechanism, I was careful to keep...

...transactivator for transcriptional activation from the 5’ LTR) and Rev (facilitates nuclear export of transcripts... is eliminated through the addition of a chimeric 5’ LTR containing a Tat-independent promoter. Although...PMID: 25075903. PubMed Central PMCID: PMC4486245. 5. Patient monitoring and follow-up in lentiviral clinical...gene therapy applications. Read on to learn more about the current (and future) applications of lentiviral...do both, Addgene depositor Didier Trono and collaborators turned to the lentivirus HIV-1, well-known to...Libraries Enable Genome-Scale, Knockout Screening Read about Genome-Wide Screening Using CRISPR/Cas9 Additional...Addgene.org Check out Our Lentivirus Guide Learn about Virus Biosafety Find Lentiviral Vectors for Your...

... cleaves in a staggered fashion, creating a 5 nucleotide 5’ overhang 18-23 bases away from the PAM. With...PAM sequence requirements. Cpf1’s preferred PAM is 5’-TTN, differing from that of Cas9 (3’-NGG) in both...genome editing than Cas9 - read on to learn more about Cpf1 and check out our CRISPR guide for a refresher...mediated cleavage are 42-44 nucleotides in length, about the same size as Cas9’s, but with the direct repeat... Cpf1 was first identified, we didn't know much about its on-target and off-target editing efficiency....CRISPR system proved we had a lot more to learn about CRISPR biology. Later work, like the adaptation ...

...circumstance. For a more detailed discussion see [5]. Problems 1 and 2 depend on protein concentrations...PMID: 17586775. PubMed Central PMCID: PMC2206698. 5. Costantini, L.M., et al., Assessing the tendency ...oligomerize under physiologic conditions. Traffic, 2012. 13(5): p. 643-9. PubMed PMID: 22289035. PubMed Central ...sold by Clontech and is in a freezer box in just about every lab in the world, is not inert. In all seriousness....11 mM [2]. Following the simple logic outlined above, you might think EGFP is unlikely to form dimers...affinity assays used in the papers describing the FPs above including molecular sizing columns and native gels...

...37 +/- 10% AAV.PHP.eB 69 +/- 4% 55 +/- 7% 76 +/- 5% Table 1: Transduction efficiency of AAV-PHP.B ...cell-type-restricted expression in both the CNS (figure 5 in Chan et al) and the PNS (supplemental figure 8 ...neurons, no transgenic mouse required (see figure 5 and supplemental figure 8 in Chan et al).  References...or the PNS, respectively.  Read on to learn more about these two new AAV capsids and how they can be useful...Important considerations when using AAVs Learn more about using AAV as a tool for gene expression in mammals...

...detection of ~5%. This means they do not reliably detect editing events that occur in less that 5% of the population...Disadvantages Mismatch Cleavage Assay Not Provided 5% Inexpensive, simple Low-throughput, low sensitivity...: 182-188. PubMed PMID: 22963480. PubMed Central. 5. Tsiatis, Athanasios C., et al. "Comparison of Sanger...et al. 2015). Meanwhile, Sanger sequencing is laborious, time consuming and cannot be applied to heterogenous...viable options for multiplex sequencing in many laboratories. Nevertheless, unbiased detection isn’t as sensitive...unbiased sequencing may not be worth the cost and labor when single clones are selected. Conversely, translational... Hough is a Biochemistry PhD candidate in the laboratory of Prof Steve Jackson at the Gurdon Institute...

...10983732. 5. Gene Therapy Net Lentiviral Vectors Page Additional Resources on the Addgene Blog 5 Tips for...vector genome which was composed of the wild-type 5’ and 3’ LTRs, the ψ sequence, a part of the env gene... from a separate plasmid, and the promoter of the 5’LTR has been deleted to reduce its activity. A CMV...first one and can be used routinely in a research laboratory. The risk of generating RCL is low but extra ...even your own! You should therefore be cautious about inserting potentially harmful genes into a transfer...

...building relationships and thinking about future career options. 5. Write a grant - even if you don’t ...mentoring group (see above). However, now that we are all so good with video calls, how about trying your hand...blog or podcast, initiated and maintained a collaborative project, served on the board of a professional...appointments with people all over the world. Think about contacting alumni of your lab or university or use...suggest you speak with. See this post that’s all about informational interviews, for more details on how...