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...) 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...

...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...

...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...

...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...

...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...

...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 ...

...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...

...consider spectral overlap. Fluorophores signal are actually quite broad, resulting in spectral overlap,...This information is important, and you’ll need to track it throughout your experiments.  Now that that’s...inhibits HRP. In those cases, thimerosal at a concentration of 0.01% w/v can be used as an alternative antimicrobial...you’ll want to pick fluorophores with the least spectral overlap between them whenever possible. This will...Fluorochromes-Brightness-Chart.pdf Syracuse Flow Core. Syracuse flow core spectral overlap. Syracuse University Flow Core Facility...January 31, 2022, from https://flowcore.syr.edu/help/spectral-overlap/   More resources on Addgene’s blog ...

...initiate a cascade that helps attack extracellular pathogens.  Transportation of antibodies to places they can...new Isotype trading cards!     References and resources References Janeway CA Jr, Travers P, Walport...region, can be bound by special receptors that transport antibodies through cells and into different body... binding capacity or valency of 10 antigens. An extra constant domain replaces the hinge region in IgM...www.ncbi.nlm.nih.gov/books/NBK27106/  Janeway CA Jr, Travers P, Walport M, et al. Immunobiology: The Immune ...

... 49,000 times! Their long-lasting popularity illustrates the utility these tools have in many different...pSpCas9(BB)). The 2A-GFP allows for screening of transfected cells. Find pSpCas9(BB)-2A-GFP (PX458). lentiCRISPR...lentiCRISPR v2 - This 3rd generation lentiviral transfer vector also from the Zhang lab is used to deliver... David Root lab is a 3rd generation lentiviral transfer plasmid for cloning and expressing new shRNA sequences...the necessary elements for packaging, reverse transcription, and integration needed for the production of... of shRNA in target cells by the addition of tetracycline/doxycycline to the grown media resulting in ...changed to clarify that it does not contain the trademarked Tet-On(R) sold by Clontech. For additional information...

...with practice. You can bootstrap your science communication skills with training programs, which are increasingly... by collaborating as they craft sentences for abstracts and presentation slides, learn from audiences ... draw and the perspective I want them to form? Training in science communication should prepare you to... of charge to accepted students, and we pay for travel expenses to our flagship workshop. In our view,...with ComSciCon, find us on Twitter, Facebook, Instragram, or send an email to [email protected]. Many...strength of story telling Science communication: 9 strategies to get your foot in the door Resources on Addgene.org...

...every diagram portraying some aspect of eukaryotic DNA would become busy and potentially detract from the ...times have you looked at a diagram depicting transcription, or DNA repair, or replication, or any number...dimensional, as you can see by this post’s chromatin illustrations from Leah Bury of Microscopic Art. I started...researching enhancer elements that control the transcription of a gene a megabase away, or looking at the...the histone methylation status at a double-strand break that needs repairing. They may be looking at chromatin... protein ZMYND8 in chromatin recognition of transcription-associated DNA damage that promotes homologous...

...proper training and guidance to the people working in the lab facility. This can be done by training eligible...and streptomycin. Mycoplasma can pass through filtration methods because of its ability to change shape...culture through various sources that are difficult to trace. These include the laboratory personnel, the serum...can spread through this form of powdered media. Filtration does not ensure complete sterilization as mycoplasma...mycoplasmas, contamination by other bacteria or extracellular fluorescent signals produced by these bacteria...rather than using 0.2-micron filters, for media filtration. There is no 100% assurance that this process...fluoroquinolone ciprofloxacin, ciprobay, zagam, baytril, tetracycline, etc. are available for mycoplasma removal from...

...assays such as mRNA data from transcriptomics; independent antibody strategies that compare the staining ...antibody that is directly conjugated to a detection substrate, such as a fluorophore, or a conjugated secondary...antibody validation.  The 5 pillars include genetic strategies that test expression in knock-out or knock-down...knock-down down tissues and cell lines; orthogonal strategies that compare antibody assay results with those...antibodies against the same target; tagged protein strategies that express a tagged version of the antigen ...antibody; and immunoprecipitation-mass spectrometry strategies that isolate an antibody’s immune complexes and...

... production and storage, we grow a little extra and transfer 100 µl of glycerol stock to each of 2 96-...through our deposit process, and then we carefully track samples through our lab using barcoded tubes and...such as special growth instructions (temperature, strain, media supplements), suggested sequencing primers...plasmid sequencing If you’re like us, you need a strategy for processing several sequencing reactions in... have different requirements for volume and concentration, and not all providers are equipped to perform...Your Plasmid Learn How Addgene Uses Barcodes to Track Samples Resources on the Addgene Website View ...