...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...

...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...

... 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...

...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...

...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...

... This is the first in a 5 part series on Management for scientists.  “I'm slowly becoming a convert ...carrot, and not the stick.     This is the first in a 5 part series on Introduction to Managing People for...the same as not being a bad manager (see cartoon above – what type of bad manager have you had?). You can...Managing scientists I should add a few special words about managing scientists because, let’s face it, we are...

...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...

...a free platform for sharing short, 3 to 5 minute videos about research. Videos can be shared by research... for communicating research. Short videos of 3 to 5 minutes provide more than enough time for researchers...measure the impact of their contribution. It offers a 5-step process and several examples for creating effective...learn about how researchers are using “big data” to look at Lyme Disease in new ways. Or learn about the...ot­her disciplines, are even less likely to read about our work. We therefore must discover new ways to.... Only on occasion do we then have time to read about research from other disciplines, or from other countries...communication through video     Video abstracts about research, especially published research, have grown...

...   The Mello lab also reported that modifying the 5’ modification of oligos used to generate linear dsDNA... animal.  PAM sequence considerations For changes 5’ to the PAM, sequences corresponding to the same strand... using two Cas9 targets. An orientation where the 5’ ends of the PAMs are facing outwards, ensuring the...Edraki A, Watts JK, Sontheimer EJ, Mello CC (2018) 5′ Modifications Improve Potency and Efficacy of DNA...learn more about genome editing with CRISPRs? Check-out Addgene's CRISPR Resources Learn about gRNA design...interest, I encountered low efficiency (0.13%) and laborious handling (screening 768 F1 animals). Recovering...

...the success of precise change via HDR limited to 0-5%. This blog post covers a brief review of different...SpCas9 rAPOBEC1 NGG 6 SpCas9 Target-AID (PmCDA1) NGG 5 SpCas9-VQR rAPOBEC1 NGA 8 SaCas9-KKH rAPOBEC1 ...PMID: 25733849. PubMed Central PMCID: PMC4371917. 5. Shimatani, Zenpei, et al. "Targeted base editing ...gene, Xw, except for a single base. If you heard about CRISPR, one question probably comes to mind: can...compatibility All of the base editors mentioned above are SpCas9 nickase-based and depend on the presence... HDR methods for rice crop improvement in the laboratory of Yinong Yang at PSU.   References 1. Komor,...The CRISPR-Cas Revolution, Cold Spring Harbor Laboratory (2018). Additional resources on the Addgene blog...

...Cellular Biology 5(11): 3274–79. PubMed PMID: 2427927. PubMed Central PMCID: PMC369144. 5. Kranz, Andrea...Cellular Level in the Mouse.” Nature Biotechnology 21(5): 562–65. PubMed PMID: 12665802. 9. Villa, Katherine... on the Addgene Blog Learn More about FLEX-vectors Learn More about Cre-Lox Check out Retrograde AAVs...expression have been developed. Read on to learn more about FLP-FRT, Cre-lox, and how combinations of FLP and...

...their Roller phenotype. If SEC is inserted at the 5’ end of a gene, the SEC separates the targeted gene...are then heat shocked to remove the SEC and after 5-6 days of growth, wild-type animals that have undergone...just one injection step! The entire protocol takes about 2-3 weeks but requires less than eight hours worth... this species, namely, the robust HDR mentioned above, an accessible germline, and multiple selectable...modular system that requires very little hands-on labor and could facilitate the tagging of every protein...

...pCL-CTIG The inducible cassette contains tTA at its 5' end and an inducible promoter at its 3' end, which... or shRNA; see article for detailed information about cloning and additional plasmids tetR-KRAB On Aebischer...

... for targeting vector assembly, and the remaining 5 plasmids include Cre and FLP expression vectors, a.... Read our CRISPR 101 post for more information about HDR/repair template design. How to use CRISPR to...

...the protein with enterokinase, which recognizes the 5 amino acids on the C terminal of the tag. Of the most... most common tags, FLAG is the most charged, with 5 negatively charged and 2 positively charged amino ...Tags have a lot of major benefits – as discussed above – but there are a few instances when they should... hindrances, and destabilization can be brought about by the tag so it’s important to validate tagged ...