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...in 5’ four base overhangs after cutting. The destination vector receives the assembly after similar Type...re-digested and "eligible” for ligation into the destination construct. Reactions are often cycled between...acquisition of antibiotic selectable markers by the destination plasmid. Antibiotic selection allowed for high...very occasional uncut or cut/re-ligated pGGA destination plasmids. A final validation of the 5-, 12- or...is dependent on the presence of every module, destination construct and functioning Type IIS restriction...

...discuss the work, set expectations, answer any questions, and (most importantly) decide on a timeline. ...regularly, make yourself available, and encourage questions. Unless you’re delegating a recurring task to ...to a seasoned report, there should be questions! Remember that delegating is not just a way to create more...team members by giving them new opportunities. I still need to check myself sometimes to make sure I am...major problem. But I also learned to ask specific questions. “How’s everything going?” is likely to get you...

...protocol for your pooled library deposit. When a requesting scientist receives a pooled library from Addgene...colonies, you’ll likely rely on serial dilutions to estimate transformation efficiency (Figure 2).    ... Figure 2: Example of a serial dilution to estimate transformation efficiency. Figure used with permission...protocol paper by the Zhang lab, and the Broad Institute’s general pDNA Library Amplification protocol are...depositing it with Addgene. If you have any specific questions about designing your library amplification protocol...

...other split systems where light is required to reconstitute enzymatic activity. Photoactivatable genome ...(Kawano et al., 2015). Nicknamed paCas9-1 and consisting of the fusion proteins N713-pMag and nMagHigh1...far-red light-activated split-Cas9 (FAST) system constitutively expresses the C-terminal fragment of Cas9 while...Michael Lin, who developed a chimeric protein consisting of Cas9 and a photo-dissociable dimeric green... prevent DNA binding in the dark. Upon light stimulation, the pdDronpa moieties move apart, allowing DNA...LOV2 has a C-terminal helix that unfolds when stimulated by blue light. When fused to the catalytic site...

...student at Stanford University. Arabidopsis is a fantastic model organism for many reasons, not the least...Agrobacterium tDNA insertion likely precipitated domestication of sweet potatoes (Kyndt et al., 2015)! It sounds...too old to transform. This transformation could still work, but the efficiency will be low. Tips: Use...plants: If you transform early, your plants will still have plenty of time to produce seeds after you expose... plant’s flowers inside. 50 mL falcon tubes or plastic weigh boats are both great options for this, depending...

...inactivation of the vector can still occur even in the case of no previous immunity existing for that adenoviral ...challenges remain (Wold & Toth, 2013).  While the pre-existing toolbox for adenoviral vectors in Addgene’s repository... – and we hope that it inspires further use of existing vectors, as well as research into the development...M. (1999). Ovine Adenovirus Vectors Overcome Preexisting Humoral Immunity against Human Adenoviruses In...

...phenotypes. Two labs, the Deverman lab at the Broad Institute and the Wilson lab at the University of Pennsylvania...the brains of C57BL/6J mice and BALB/cJ mice, suggesting it was a likely player in PHP.B permissiveness...receptor (AAVR). Cell lines lacking galactose were still transduced by PHP.B but only if Ly6a was expressed...with PHP.eB, an enhanced version of PHP.B, virus still reached the brain endothelial cells of these mice... available from Addgene. References Holmes, Christina, and William L. Stanford. "Concise review: stem...

...was applying. I probably did not respond to her question as well then as I would now. I would have to repress... repress laughter if I was asked that question today, because I sometimes wish for a stretch of time that...me, “I find that at a desk, it is much easier to stick to timelines. This is probably because you don't...careers and desk jobs “alternative careers.” Realistically, based on the numbers of PhDs and available ...into science in the first place. Finally, if you still find the idea of not holding a pipette again upsetting...

...synthesis (those of us who have used antibodies for diagnostics know how frustrating it can be to get a “bad ...the bound protein fluorophores and quenchers are still relatively bulky. One of the advantages of oligonucleotides...stable and brighter version of the aptamer, but it still had limitations in vivo. Using a combination of ... a lower magnesium dependence. Though initially still unstable in eukaryotes, the Jaffrey lab has since...et al. “Aptamer-based assay for prion diseases diagnostic”. Procedia Environmental Sciences. Volume 12,...

...full length gene and/or protein is reconstituted. One way to reconstitute a split protein like Cas9 is to...allow for more room for regulatory sequences while still fitting Cas9 and its gRNAs into one AAV, Cas9 must...confirming its suitability for in vivo studies. Testing AAV-SaCas9 in vivo To test the efficiency of AAV-SaCas9...Nogales E, Doudna JA (2019) CasX enzymes comprise a distinct family of RNA-guided genome editors. Nature 566...

... 1–9 (2017).Walton, R. T., Christie, K. A., Whittaker, M. N. & Kleinstiver, B. P. Unconstrained genome... effects. The SpG variant, discovered by the Kleinstiver group has a PAM of NGN (Walton et al., 2020)....Don’t want to permanently modify the genome but still want to affect gene expression? dCas9 can be used... and antibody-activator fusion proteins. The Synergistic Activation Mediator complex, generally the strongest...

..., black pellet was sent to the AbuGoot lab for testing, they weren’t sure what they would find… but it...new CRISPR effector protein they named Cas7-11. Testing showed that Cas7-11 cut RNA with unprecedented ...insertions, and deletions. But CRISPR’s uses were still limited to small deletions and small insertions,...that gap. Consider, for instance, a disease like cystic fibrosis, which can be caused by thousands of different... Yarnall, Cian Schmitt-Ulms, Rohan N. Krajeski, Justin Lim, Lukas Villiger, Wenyuan Zhou, Kaiyi Jiang,...

...range of logistical barriers. Addgene worked to identify, reduce, and remove these logistical barriers...for people depositing plasmids and for people requesting them. Prior to this, many plasmids were available...packages navigated customs and arrived at their destination in a timely manner and at the correct temperature...to scientists across the globe?” By asking this question, and by listening to the answers from our community...

... during the early stages of the process. Some questions to start with are: Is your sequence of interest...opposite (reverse) orientation? The answers to questions like these will determine which acceptor vectors...assembly efficiency. This can be done through domestication, which is the modification of a DNA sequence... the synonymous mutation of a codon. You can domesticate the sites yourself using PCR, but gene synthesis...gene synthesis, you can request the sites to be domesticated through the process, and request your sequence...

... plasmid is delivered to your target cells, you still need to select for the cells that contain it. Antibiotic... approach in a mammalian cell experiment should still include an antibiotic resistance gene for growth...express your protein of interest. However, you do still need a species-compatible promoter for expressing... consider the viral vectors themselves to be a distinct gene delivery method. We focused this post on ...between prokaryotes and eukaryotes. Conclusion I still think it’s pretty incredible that my cells “speak...

...the way we apprehend molecular complexes and is still a very useful tool.    Like a guitar hero (that ...SYFP2A, mTurquoise/mVenus and others. CFP-YFP is still one of the best and most used pairs to measure FRET... is difficult to address especially when you investigate unknown molecular complexes. But it is a parameter... faster measurement systems, this technique has still many good days and good “gigs” ahead...No offense...Chemiluminescence Resonance Energy Transfer    Interesting Articles About FRET: Piston DW, Kremers GJ. ...

...potential for therapeutic drug discovery as it is estimated that 75% of disease causing genes in humans have...Stainer group determined that there are actually two distinct populations of β-cells that originate in different...other model organisms. Worms have been used to investigate an array of biological systems and processes ...PubMed PMID: 9486653. Hesselson, Daniel, et al. "Distinct populations of quiescent and proliferative pancreatic...19706417. PubMed Central PMCID: PMC2736433. Howe, Kerstin, et al. "The zebrafish reference genome sequence...

...GFP-like probes by supplying a single gene to cells. Still in bacteria, which do not produce BV, an additional...mouse. Image courtesy of Axel Nimmerjahn (Salk Institute for Biological Studies) Multiple uses: From...applications.  There is a whole set of spectrally distinct (e)miRFPs available. The numbers in (e)miRFP names...occurring due to protein synthesis, degradation, or reconstitution (reviewed in Shcherbakova et al., 2018). For...developed based on mIFP6. Learn more about the characteristics, performance, and applications of near-infrared...

...epigenetic editor consisting of dCas9 fused to a DNA methyltransferase — that can drive lasting transcriptional...cofactor fused to a histone tail to recruit and stimulate the endogenous methyltransferase. The strategy...H3K4me0 on D3L, releasing the autoinhibition and stimulating the methylation of nearby CpG sites on the target...expression off. Plus, DNA methylation is a long-lasting repressive marker, allowing the team to develop...

...article is to provide enough background for you to distinguish the features of any common lab strain and determine...them in 1885. E. coli are mainly found in the intestinal tract of animals. There are many different naturally...hsdR(rk-, mk+) Unmethylated DNA not degraded, cell still can methylate DNA   hsdS(rk-,mk-) Unmethylated... Hte   High transformation efficiency deoR constitutive expression of genes for deoxyribose synthesis...strains; however, these tables are by no means exhaustive! For a more comprehensive list and additional...