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...two posts in this series described a practical approach to selecting a bright fluorescent protein and ...aggregates. This property can be tolerated in some applications (e.g. labeling of cells or tracking promotor...promotor activity), but it is problematic in applications in which the fluorescent protein is used as an inert...cells. To understand how well fluorescent proteins approach this ideal situation, cellular assays have been...can be used in the OSER assay including CytERM-mKOkappa, CytERM-mTurquoise2, and CytERM-dTomato. When...

...DNA-binding proteins to label certain loci, but this approach is not scalable for every locus...unlike CRISPR... 9 and 13. They next turned their attention to mapping pairs of intrachromosomal loci. The technique successfully...authors’ knowledge, this work represents the first mapping of intrachromosomal loci, a major benchmark in ...-MS2-PP7 Despite the success of Ma et al.’s approach, fluorescently labeled Cas9’s come with a number...up fluorescent CRISPR labeling, they took a new approach: labeling the gRNA itself. In their CRISPRainbow...

...regions, both through genetic and proteolytic approaches. This comes in handy when, for example, you want... as secondary antibodies, have much broader applicability. It is fairly easy to find these in a Fab fragment...pre-form primary-secondary antibody complexes before applying them to samples, such as when performing IHC in...can be used as a block in similar scenarios. By applying unconjugated Fab fragments that are the same species...fragments described above can be used for similar applications and have similar advantages, though with some...

...engineering fields. We’ll walk you through how to apply this precise and easy-to-use system to your cloning...designed correctly, the recognition sites do not appear in the final construct, allowing for precise, scarless...are eliminated by digestion/ligation and do not appear in the final construct. The destination vector ...adapted Golden Gate cloning for inserting the appropriate oligonucleotides specifying a gRNA target sequence...different combinations, or other new and exciting applications, this system is an incredibly powerful tool ...

...end-users, size of the transgene, and downstream applications should all be considered when choosing the correct...example, in our hands we find that a double harvest approach at 48 and 72h produces high titer virus for transfer...procedure for a single 10 cm2 dish can be easily applied to multiple dishes, remember that handling multiple... as resources available, cost and downstream applications. For example, while the PEG precipitation method... When assessing lentiviral titer, don't compare apples to oranges After choosing a packaging system and...

...information to Addgene Material Transfer Agreement (MTA) approval  Send plasmids to Addgene  Quality control  In...can also directly paste sequence data into the appropriate full and partial sequence cells. *Note: If you...descriptive plasmid name and match the name as it appears in its publication or as it is commonly known in...descriptive purpose focuses on the practical application of the plasmid and will help a scientist outside... account username. In order for the plasmids to appear in your Addgene account under My Plasmids, we need...

... editing in primary human cells  Cell therapy approaches can use base editors to correct mutations in ... reduced off-target editing frequencies. For applications that require high DNA editing specificity, for... for example for potential therapeutic approaches, the authors recommend mRNA delivery, the V106W version...deamination, an important attribute for therapeutic application. ABE8s demonstrate an overall improved base editing...editors with increased activity and therapeutic application. Nature Biotechnology. https://doi.org/10.1038...

...When possible, the PE3b/PE5b approach is recommended over the PE3/PE5 approach. Figure 7: Nicking...its design changes for every new prime editing application. And optimizing its sequence is important, as...with BioRender.com. PE3b and PE5b: In this approach, the nicking sgRNA is designed to bind only after...BioRender.com. MMR inhibitors PE4 and PE5: These approaches inhibit MMR to improve editing efficiency, but...

..., its dimeric nature can interfere with some applications. Several labs have been working to produce monomeric... for molecular fusion and membrane-targeting applications. Nature Methods, 1–9. https://doi.org/10.1038...successful in primates. The authors used a multispecies approach to generate and screen a library of AAV9 variants...proven itself a good option for essentially all applications. Addgene’s plasmid collection includes over ...clone (M2) has been widely used in a variety of applications. Find anti-DYKDDDDK recombinant antibodies here...

...circular assembly, with the 52-piece approach yielding approximately ~800-fold fewer plaques than the 10...thought otherwise! They suspected a data-driven approach might allow for an increase in the complexity....to sets of up to 6-8 overhangs, the data-driven approach was needed to massively expand complexity. The...Overhang Ligation Fidelity by T4 DNA Ligase and Application to DNA Assembly. ACS Synthetic Biology. https...

...to take, and if something does happen, ensure you can receive appropriate treatment and/or monitoring. ...protection are key to lab safety and the same principles apply to viral handling. Gloves are always required and...start an experiment, just in case something does happen.   Finally, the best way to be safe is to know ...valuable tools without endangering yourself or others. Happy transducing! Resources Resources on Addgene.org...

.... We both had an applied science bent, so the engineering aspect of this job appealed to us. However, ...possibilities in life for employment?  It never happened – there was never a “moment”. But there were a...about continuing with the business. We ended up applying to a business accelerator freely stating that ...your thoughts with us in the comments below. We’re happy to answer questions about this transition (so far...

...carefully) holding plates up to the light, where they appear as opaque dots (colonies should contain at least...only 1.5 ml of each are used). This solution is capped, swirled and tested for the correct temperature...and not spread out onto the plate. We test the (capped) 1:1 agarose/DMEM solution against a part of the...densities, then dispose of the plates that do not appear to be yielding well-spaced clones. For rapidly ...population of transfected clones. The technique is applicable to a wide variety of cell lines, from HEK to ...

...contain rAAV. To minimize contamination, use appropriate decontamination procedures (Korte et al., 2021...surfaces that come into contact with the rAAV and apply common cell culture practices diligently throughout... toxic transfection reagents and keep the cells happy. Harvesting virus Once you have transfected your...al., 2010). For certain in vitro and in vivo applications, you can consider using unpurified AAV from ..., 2017). You can learn more about common AAV applications by reading our Viral Vectors 101 series!  This...

...uncontrolled cellular proliferation. We have appreciated for some time the causes of this uncontrolled...cellular machinery necessary for division. This approach also kills dividing non-cancerous cells, such ... University of California, San Francisco, is approaching the Ras problem by exploiting the different mutant...genes from the Ras signaling pathway. Its major appeal is that it enables the simple construction of vectors...any of these genes through several different applications such as fluorescent protein fusion studies and...

...been excitedly testing NgAgo's genome editing applications. So, how does NgAgo stack up thus far? A few...tips. Based on the discussions in the forums, it appeared that many researchers were having difficulty achieving... target site. Overall, 64% of researchers were unhappy with the new method and 65% wanted to wait until... the comments section below. Keep pipetting and happy genome editing! Check Out Other Argonaute Plasmids... S. Lander, and Feng Zhang. "Development and applications of CRISPR-Cas9 for genome engineering." Cell...

...in the limelight. On December 19, 2017 the FDA approved Luxturna™ the first gene therapy for the treatment...AAV-based gene therapy treatment for a genetic disease approved by the FDA is for a rare eye disease and not another...trial started, and 10 more years to obtain FDA approval and make history in the form of Luxturna™. A ...medicine. References and further reading 1. FDA approves gene therapy for blindness 2. Gene Therapy emerges...-1250015778 7. Yu W and Wu Z., 2018. In vivo Applications of CRISPR-Based Genome Editing in the Retina...

...lot of activities (try for a dozen!). The best approach may not be “let’s try to make this STEM activity...that would compliment the STEM curriculum. As it happened, I’d met some of the 8th grade science teachers...opinion, it was more important to communicate the applications of CRISPR over the details of how it worked....analogy that involves student participation, make it happen! How should we use CRISPR? How shouldn’t we use... I came out of the experience with a renewed appreciation of our science teachers’ difficult-but-rewarding...

...variations in gene expression, they found that for approximately 40% of the identified genes, the genes were ...lines from individuals of African descent have approximately 20% more single nucleotide polymorphisms (SNPs...ancestry-associated cancer dependencies are undercounted by approximately 20%. In some cases, this may lead researchers...map. Now, any data that came from CRISPR guides mapped to a SNP would be ignored. Working with Lia Petronio...8 Lewis, Anna CF, Santiago J. Molina, Paul S. Appelbaum, Bege Dauda, Anna Di Rienzo, Agustin Fuentes, ...

...common antibiotic resistance genes and which applications are best (or worst) for each antibiotic. The... after it has been heat-shocked or electrically zapped. As a result, a shorter recovery period of 30 minutes...is expensive (relative to amp), and for some applications, the benefit it offers is marginal. Fortunately...antibiotic resistance ultimately comes down to your applications and needs. The more niche and specific your ...recap of the pros and cons of each antibiotic and happy selecting!   Antibiotic Resistance Stability...