...their scientific careers hoping to follow the traditional academic route (find tips for getting a faculty...comprehensive lab experience. With this valuable training behind you (and largely only that is necessary...from around the world easier. You should also demonstrate a strong willingness to help others with their...attitude, and an open-minded personality. These traits will help you deal with any incoming requests or... get involved in product testing in the lab, to travel to customers’ sites, and to run experiments with...actually caused the problem. It may not always be straightforward to identify those crucial details because there... The benefit is that these skills are highly transferable and will help you enter other roles in project...

...been significant work on constructing Cas9 transcriptional activators. These constructs allow for the ...Cas9 deficient in DNA cutting activity to a transcriptional activation domain (Fig 1). Two ways to use...promoter of another gene, thereby driving aberrant transcription. That being said, we generally pick guides by...picking whichever guides are closest to the transcription start site (TSS). We recommend targeting the...Latency Reversal Using CRISPR/Cas9-Derived Transcriptional Activator Systems. PLoS ONE 11:e0158294 . https...Church GM (2015) Highly efficient Cas9-mediated transcriptional programming. Nat Methods 12:326–328 . https...Nishimasu H, Nureki O, Zhang F (2014) Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex...

...plasmid post transfection. The 2A "self cleaving" peptide causes the ribosome to translate Cas9-2A and ...be used to transfect a variety of mammalian cells and can be modified for viral trasduction (see the Zhang...component of most CRISPR systems and generates double strange breaks at target sites 5' of the 5'NGG3' PAM sequence...can be used to test for SpCas9 expression post transfection by western blot. Overall, this is a versatile...2013): 2281-2308. PubMed PMID: 24157548. PubMed Central PMCID: PMC3969860. Resources on the Addgene Blog...

...possible to multiplex ultrasound dyes because these dyes “collapse” at different ultrasound frequencies. Read...non-invasive methods for cellular imaging with ultrasound. In his research he applies naturally occurring...of dyes allows dye visualization in vivo using ultrasound (for an example see the figure below). In addition...addition to low costs, deep tissue penetration, and high spatial resolution are benefits of this technique...Read more about ‘Using ultrasound to image bacteria in vivo: acoustic reporter genes’ on our blog. Creative....7686 (2018): 86.PubMed PMID: 29300010. PubMed Central PMCID: PMC5920530.  Resources on the Addgene Blog...

...Uniform Biological Material Transfer Agreement (UBMTA) for its material transfers. The UBMTA was drafted by... has time for paperwork? Here’s why a Material Transfer Agreement (MTA) matters: Science thrives on collaboration...universities and institutions require them for material transfers. When Addgene entered the plasmid sharing space...order, Addgene generates the MTA and emails it straight to you, or we can send it directly to your institution...receiving institution. Therefore, many technology transfer officers are familiar with the UBMTA and are happy...  material transfer agreement...

...systems Name Function Plasmid Transduction Marker Mouse Strain C-CHIME Knocks out a pair of... LaFleur et al., 2019b). CHIME works through transduction of Cas9-expressing (Rosa26-Cas9) hematopoietic... a lentiviral vector containing a gRNA. These transduced stem cells can be implanted in irradiated recipients...suite of four systems which utilizes custom mouse strains and lentiviral expression vectors (Table 1). The...positive control genes in CD4+ and CD8+ T cells. To demonstrate the power of these systems to investigate in ...generation of exhausted CD8+ T cell subpopulations and restrains tumor immunity. Nature Immunology 20, 1335–1347...

... that we can invest our time into learning and extract the most value? This is only one of many decisions...tools assess repair events by type (NHEJ/HDR) and track indels at a single locus, usually the target site...exclusively for NGG PAM targets and began allowing for arbitrary PAM definitions. These newer tools may still prove...on downstream results. Examples: “HTML”, “visual track”, “plot”, “tabular”, “interactive”, and “save”. ...enthusiasts, take a look at sgRNAcas9. Top tools for transferring CRISPR technology to a new organism: For obvious...but comes with a graphical user interface. Best strategy for labs: Define what you need the software tool...on your needs. We will likely begin to see a contraction of available tools with more and more features...

...them to track and find data and information. If an employee leaves the institution, the transition is smooth...that bring more to the table in terms of data traceability and reproducibility of research. Electronic ...work. It is already becoming impossible to keep track of it all in paper notebooks. Following these findings...one place, easily retrievable, searchable and traceable. However, switching from paper to an electronic...requires time and knowledge. In a recent article, we translated our knowledge of electronic lab notebooks into...electronic signatures, electronic witnessing and audit trails. How does the ELN provider protect and respect...Researchers spend 40% of their time on tedious administrative tasks that drain resources from the actual ...

...) to remove mutated exons from the dystrophin transcript. Unfortunately, the oligonucleotides only modestly... DMD patients. Figure 1: Exon skipping strategies for Duchenne muscular dystrophy. gRNAs can be...in one experiment, they found the exon-skipped transcript constituted 59% of total dystrophin mRNA, similar...human-relevant exons 45-55, rather than in the traditional exon 23-mutated mouse model. Potential off-target...Feb;24(1):69-86. PubMed PMID: 24506781. PubMed Central PMCID: PMC3922311. 6. Tabebordbar M, Zhu K, Cheng...Mar;23(3):523-32. PubMed PMID: 25492562. PubMed Central PMCID: PMC4351462. 8. Ousterout DG, Kabadi AM, ...2015b Feb 18;6:6244. PubMed PMID: 25692716. PubMed Central PMCID: PMC4335351. 9. Ran FA, Cong L, Yan WX, Scott...

...bound to anything? Is it a transmembrane protein, or is it know to translocate or fold in certain conditions...as part of performing a specific function. A transmembrane protein that is being used as a marker for flow...out of a complex mix of proteins that have been extracted from a cell, instead of when it is attached to...looking for a protein of interest while it’s in a transition state or specific configuration. So how do you...conditions? It can help to visualize an abstract protein during your sample prep process, imagining how it...the assay can help you avoid a great deal of frustration when designing and troubleshooting immunoimaging...

...barcodes within the gRNA sequences. This allowed tracing of the performance of each gRNA multiple times ...using manipulations such as injection of precise titrations of AAV or heat-shocking the organism. The SPARC...protein phosphatase that responds to rising intracellular calcium levels within the cell. When levels ...required for virulence in pathogenic fungi, it is an attractive drug target. Calcineurin reporters allow scientists...Sabrina Büttner’s lab designed a calcineurin transcriptional reporter consisting of a quickly-degraded destabilized... wild type (top panels) and cells with high intracellular Ca2+ (bottom panels) equipped with destabilized...contributed by Jennifer Tsang Figure 4: Double stranded DNA bound by tTALE-FP with the fluorescent protein...

...repair templates. Their work demonstrates that different repair template strategies activate different HDR ...sub-pathways like synthesis-dependent strand annealing (SDSA) and single-strand DNA incorporation (ssDI). "Easi-CRISPR...These complementary goals are aimed at improving translation of CRISPR technology, but they would also bring...a DNA repair newbie - diagrams of resection and strand invasion are like a foreign language to me. But...(2017):1099-1111. PubMed PMID: 28356322 PubMed Central PMCID: PMC5495063 Kan et al. use a GFP-BFP conversion...

...relatively straightforward to define, but for control elements such as promoters and transcription terminators...viral strains. We are working on a way for SnapGene users to apply features from a reference strain to newly.... Preventable errors in the design of cloning strategies set experiments back days or even weeks. Primer... and the use of genes from different microbial strains. It proved to be impractical to catalog every variant... newly sequenced strains through our mismatch tolerant detection algorithm and thereby speed up the annotation...

...monoclonal antibodies. The good news: there are strategies for mitigating this issue, and we’ve highlighted...staining by the secondary, then the following three strategies might help!     Figure 1: Species on ...in the tissue. Created with biorender.com. Strategies for avoiding background secondary binding Direct...Goodpaster, et al., 2013). One issue with this strategy is that large complexes may not permeate through...(Figure 2C) Speaking of Fab fragments… Another strategy for species-on-species IHC protocols is to block...solutions will help get your experiments back on track.  One final note — this post focuses on the species-on-species...) A novel immunohistochemical technique for demonstration of specific binding of human monoclonal antibodies...

...1970s by Alex van der Eb in the Netherlands. Transformation of HEK cells with adenovirus 5 DNA was postdoc...with kidney cells (Shaw et al.). Since then, transcriptome studies of the cell line have determined it ...replication. To generate a virus, you need only transfect the ‘helper’ plasmids (viral packaging machinery...cell or organism. This pipeline is utilized to transduce cell lines for basic science research all the ...Protein production  HEKs are extremely easy to transfect in bulk and produce recombinant proteins at high...widely available, which can make for a seamless transition of biological tools to other researchers using...Resources References Shaw G., et al., Preferential transformation of human neuronal cells by human adenoviruses...

...guides will also need to be transfected into the cells of interest. In contrast, RNA guides must be expressed...NgAgo ARTICLE DISCUSSED IN THIS POST HAS BEEN RETRACTED AND FOLLOW UP STUDIES HAVE FAILED TO REPEAT THE...expressed from a plasmid or in vitro transcribed, and changes in gRNA secondary structure can affect editing...removal RNA-guided CRISPR enzymes cause double-stranded breaks, with either blunt (Cas9) or staggered ...) (2014):258-61. PubMed PMID: 24531762. PubMed Central PMCID: PMC4697943. Find plasmids from this publication...) (2015):5120-9. PubMed PMID: 25925567. PubMed Central PMCID: PMC4446448. Additional Resources on the ...

...used a transgene selection strategy for their C. elegans/CRISPR experiments, but this strategy required...can generate a fluorescent protein fusion, transcriptional reporter, and loss-of-function allele in just... the gene of interest. L1/L2 larvae from these strains are then heat shocked to remove the SEC and after...Figure 2: Schematic from Dickinson et al. (2015) illustrating the steps to fluorescently tag a C. elegans ...plasmids from this publication at Addgene. Other strategies for CRISPR/Cas9 editing in C. elegans: Engineering...

...platform for pre-registration wherein researchers can submit designs and transparently reveal them upon... characterised antibodies against all human extracellular proteins with community-based validation. They...obvious solution to this problem would be for traditional journals to publish negative data, but this is...publicise negative data - we don’t have to rely on traditional journals. There’s social media - science Twitter...review our experimental designs and we need to be transparent about the reasons we alter these designs if we... effective if it were more practical) is pre-registration and review of experimental design as part of...your eyes, but even if this ideal form of pre-registration doesn’t come to fruition any time soon, the ...

...using an epitope-based strategy such as Anti-6xHis. You could also consider strategies using other types of...similar to standard immunofluorescence, but with some extra considerations (Im et al., 2019; McLaughlin, 2019...antibodies from Addgene), or be prepared to run some extra controls and validate the antibody yourself. If ...anti-Rabbit IgG   If you need more signal, another strategy is to use a biotinylated secondary antibody then...washed away and leave no signal. If you see lots of extra signal in these samples where you don’t expect to...you may need to optimize the conditions and concentrations of all the different antibodies, blocking, ..., Manning, C. F., Nguyen, J. T., Bechtold, H., Dumitras, C., Gong, B., Nguyen, K., Van Der List, D., Murray...

...fluorescent protein transcription in response to river contaminants and transform these plasmids into ...into a bacterial strain. The sensor will be transported inside a small 3D printed robot that will “swim” ...Klebsiella and Pseudomonas isolates causing urinary tract infections. This project aims to examine the antibiotic...fluorescent proteins will be used to tag individual strains of bacteria. Their interactions and growth within...research related to space medicine. Now he aims on tracking pollution in rivers by employing synthetic biology...