...gram-negative bacterial endotoxin is ensured to be less than 5 endotoxin units per mL. The endotoxin levels are determined... before being distributed to customers. Details about our production protocols, titering methods, and ...either in-house by Addgene scientists or through collaboration with viral vector manufacturing facilities, ...

...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...

... Cell 18, no. 5 (2016): 573-86. PubMed PMID: 27152442. PubMed Central PMCID: 4871596.  5. González, Federico...phosphatase treatment, substituting cytidine with 5-methylcytidine, and/or substituting uridine with pseudouridine...by the piggyBac transposon." Nature Methods 6, no. 5 (2009): 363-69. PubMed PMID: 19337237. PubMed Central...PubMed PMID: 20952534. PubMed Central PMCID: 2956203 5. Robinton, Daisy A., and George Q. Daley. "The promise...methods do you have experience using? Let us know about your iPSCs experiences in the comments below! References...Additional Resources on the Addgene Blog Learn about Methods to Reprogram Peripheral Blood Cells Check...

...This is the third in a 5 part series on Management for scientists. Feedback is help and it should result...build it into your routines. This is the third in a 5 part series on Management for Scientists. Subscribe...employee about what they do well and where they need to improve, and they’re also clear about how the ...enough to tell them about areas they need to improve in. If a manager has concerns about an employee and ...language to talk about real performance issues. Watch this little video to learn more about indirect language...Medical School researcher Connie Cepko to learn about her management style. Formalizing the feedback session...informal feedback is a very effective way to bring about change in behaviors and habits in a nonthreatening...

...exo-) DNA Polymerase I, Large (Klenow) Fragment (3'-5' exo-) 165512 pOpen-THEAQpolA Taq DNA Polymerase 165513...engineering and directed evolution. More information about the collection can be obtained at the Open Bioeconomy...

...Bleomycin glycopeptide Induces DNA breaks Bactericidal 5-100 ug/mL Erythromycin macrolide Blocks 50S ribosomal...subunit; inhibits peptidyl translocation Bacteriostatic 5-25 ug/mL in EtOH *In prokaryotes.     **Dissolve...molecular biology, yet scientists rarely stop to think about how much easier it makes our lives. Plasmid transformation... sterile filter unless otherwise specified. The above table lists some antibiotics commonly found in the...or light – do not add them to media hotter than about 55oC and store plates/stocks wrapped in foil if ...

...outperform Cas9 in GC-rich regions. DNA guides NgAgo uses 5’ phosphorylated DNA guides (so called “gDNAs”) rather...if NgAgo preferred certain guide sequences - using 5 guides/gene for 10 genes, they did not observe differences...impaired NgAgo activity, with three mismatches abolishing activity. One-guide faithful NgAgo guide loading... how it’s working in the comments section! Read About NgAgo User's First Impressions Here References ... Additional Resources on the Addgene Blog Read About Cas9 Homolog Cpf1 Check Out High Fidelty Cas9 Mutants...

...pLionII 3rd FIV-derived (FELIX) plasmid with a chimeric 5’LTR, Any expression cassette (promoter and gene of... Read our Lentiviral Guide for more information about: Lentiviral components 2nd versus 3rd generation...systems Viral production Frequently asked questions about lentivirus use Glossary of commonly used lentiviral... or shRNA. See article for detailed information about cloning & additional plasmids. Aebischer and Trono...

...consistent 5% positive error across the plate is fine; a 5% positive error on one side and a 5% negative...  Probe should have a melting temperature 5-10 degrees above the primer and be at least 25 bp away from... Probe is on a hairpin structure included in the 5’ primer Yes Yes. Agarose gel  LUX fluorogenic...

...Several behavioral assays can be done on zebrafish at 5 days post fertilization, including escape behavior...behaviors, the researchers measured the tail movements of 5 day old fish. The larvae were immobilized in agar ... posts about viral vectors Subscribe to the Addgene blog Resources on Addgene.org Learn about our ready-to-use...