...are many docking sites available for integration. 2. How soon would you like your flies ready for use?...beautiful immunofluorescent images like in this paper (2). Metabolic assays such as Seahorse can be employed...PMID: 16547094. PubMed Central PMCID: PMC1526508. 2. H. Jiang,  et al. Cytokine/Jak/Stat signaling mediates...Harvard University. In my previous two posts, I’ve described the fundamentals of how to work with Drosophila...Drosophila as an experimental model organism. I then described the Gal4/UAS system used by geneticists to study....e. there won’t be positioning effects). When designing experiments where you want to express multiple...wondering, “How do I know which of my flies have the desired integration?” Not to worry! In flies, phenotypic...

...required to make a plasmid. In 2023 (and at closer to 2 million plasmids shared than not!), we think it’s ...time does it take to make a plasmid? Figure 2: Survey results from a Twitter poll run in 2023, n...go wrong, replace them for free. Plasmids ship out 2-3 days after ordering, making it a toss-up as to whether...in science – the time spent by the scientists designing and performing the experiments. This cost varies...quickly with the postdoc who’s really good at primer design – but first, you have to know what resources exist...well) plate! How much do repositories help?   Designing precise calculations for any of the numbers in...engagement with and use of repositories like Addgene provides large-scale savings. How large? Using Doench’s...

...termed fluorescence activated cell sorting or FACS (2). By analyzing single cells instead of the population...expression. A routine flow cytometry experiment uses 2 or 3 different fluorescent colors each measuring a...distinct populations on a FSC versus SSC plot (Figure 2).  A user can then “gate” around the specific population...cell and T cell into distinct populations (Figure 2). An immunologist who studies B cells, might then ... B cells, plasma cells or others.   Figure 2: Due to differences in their size and complexity, ...assessed for the desired parameter. A droplet containing a cell that is positive for a desired parameter is...their observations. However, while flow cytometry provides analysis at the cellular level, it cannot be used...

...Central PMCID: PMC4151723. 2. Doench, John G., et al. "Optimized sgRNA design to maximize activity and ...CombiGEM lentiviral vector backbone pAWp12 (Figure 2). Cloning from the pAWp28 storage vector to the pAWp12... before beginning your experiments. Figure 2: Strategy for assembling barcoded combinatorial gRNA... Blog Learn Best Practices for Designing Your gRNA Read How Barcodes Can Be Used in Lineage Tracing Read...genetic perturbations. The ease with which one can design and synthesize CRISPR guide RNAs (gRNAs) for genome...their functions. The continual advancements in gRNA design necessary to achieve maximal on-target and minimal...regulation in biological systems. The CombiGEM platform provides a means to create barcoded gRNA libraries that...

...Step 2: Develop a research plan Most grant applications restrict the space available to describe the ...require application submission in-house approximately 2 weeks before the RFA due date in order for the administrative...proposal typically lists the deliverables, explains 1-2 potential pitfalls of the intended protocol, and offers...application. It typically includes overview information, key dates, funding description, research plan, award... setbacks, and potential alternative routes, Provides opportunities to convince others why a project...aspects are understood. While reading the funding description, pay attention to the keywords used and think...literature and offer educated insight into what the desirable features of future technologies should offer in...

...of other Cas proteins found in the so-called class 2 CRISPR-Cas systems. There are 24 variants of the cas14...the presence of infectious organisms (ex: SARS-CoV-2) and genetic mutations. By providing a gRNA specific... cas1, cas2, cas4, and a new gene cas14. cas14 encodes for a small Cas protein (40-70 kDa), which is half...Harrington, Lucas B., et al. "Programmed DNA destruction by miniature CRISPR-Cas14 enzymes." Science 362.6416...

...CcdB.” Biotechniques. 1996 Aug;21(2):320-3. PubMed PMID: 8862819. 2. Bahassi, EM., et al. “F plasmid CcdB...those that do not contain the desired insert should not grow.   Figure 2: Image from http://teachline.ls.huji.ac.il... plasmid maintenance during cell division. ccdB codes for the toxic protein (CcdB) that acts as a DNA ...death. ccdA, another gene found in the ccd operon, codes for the antitoxin protein (CcdA) that protects the...of ccdB, leading to positive identification of desired clones.   ...Becomes a powerful cloning tool...

... (2,3). Stable strand separation is maintained by 2 sets of interactions : 1. Interactions between the...formed by the Cas9 HNH and RuvC nuclease domains 2. Interactions between the target strand and the gRNA... Find plasmids from this publication at Addgene. 2. Nishimasu, Hiroshi, et al. "Crystal structure of ... nuclease activity of the Cas9 nuclease itself. Design and testing of eSpCas9 Looking at the structure...and the negatively charged DNA and therefore destabilize basal strand separation. This decreased strand... choice and get improved on-target specificity. Design and testing of SpCas9-HF1 Kleinstiver et al and...Joining Repairs Double Strand Breaks Get Tips on Designing Your gRNA Browse Our CRISPR 101 Series Resources...

...phosphorylation events in the proteomes of higher mammals (2). How can we begin to understand the importance of... the open reading frame (ORF) of interest (Figure 2). 1. The phosphoserine OTS The backbone of the phosphoserine...optimized Sep-OTSλ and can recommend its use (6).  2. C321, a specialized bacterial host strain for pSer-incorporation...biology4.5 (2002): E127-E130. PubMed PMID: 11988757. 2. Hornbeck, Peter V., et al. "PhosphoSitePlus, 2014...site-specific incorporation of phosphoserine (as described below) provides researchers with a valuable tool for understanding...organisms lacked a classic cysteinyl tRNA synthetase, despite abundant incorporation of cysteine in their proteomes...pSer-incorporated proteins on a single plasmid. This includes 4 copies of a tRNA that specifically recognizes...

...preventing design of antibodies specific to each fluorophore. Third, Brainbow-1 and Brainbow-2 each contained...Brainbow mouse neural labeling system. Brainbow-1 and -2 have been further refined to create Brainbow 3.2. ...M-CFP expression; these deletions are defined using 2 loxP variants (only identical loxP sites can mediate...processes not previously visible with Brainbow-1 and -2. The general structure of Brainbow-1.0 is retained...R26R-Confetti described in Hugo J. Snippert, et al. (2010) and the MAGIC Marker strategy described in Karine...Brainbow-1 system employs Cre-mediated deletion, the design of Brainbow-2.1 cleverly combines Cre-mediated ...immediately following the promoter. The STOP cassette includes a mutant YFP that does not fluoresce, but can ...

...difficult-to-manipulate primary cells, achieving knockdown within 1-2 hr of reagent delivery. TRIM-Away is suitable for ...factor 1 controls expression of transcription factor 2, which directly controls expression of a reporter ...modulating the induction of transcription factors 1 and 2. See Aranda-Díaz et al. for more information. The ...TRIM21 recruits ubiquitin and the proteasome to destroy antibody-bound pathogens. Clift et al. found that...necessary for studying endosomes, the Hell lab designed their super-resolution pH indicators (SRpHi) biosensors...sgRNAs are used with FIRE-Cas9, experiments can be designed to target multiple loci at once. In addition, ...

...Quotes taken directly from participants (summarized in 2) touched upon many problems in this area: “ [The] ...PMID: 24733905. Pubmed Central PMCID: PMC4000813. 2. McDowell, Gary S., et al. "Shaping the Future of ...A fair deal for PhD students and postdocs." Elife 2 (2013): e01139. Pubmed PMID: 24137543. Pubmed Central...growing dialogue on the state of a system that, despite all of its successes, has many deep-rooted flaws...all bad, however - there are some signs that the tides may be turning. In 2013, the NIH announced its first...in Scientific Training (BEST) awards, which are designed to support new approaches that address the need...

...calcium biosensor, so that the (1) binding of calcium, (2) allosteric rearrangements of the sensor, and (3) ...user-controlled illumination with violet light and (2) exposure to increased intracellular calcium. It has...dynamics in vivo. Neuroscience Research. 2021 Aug 1;169:2-8. Suzuki J, Kanemaru K, Iino M. Genetically encoded... There are several calcium reporters specially designed for the aims and requirements of in vivo studies...populations of interest. Many of the calcium sensors described are also already packaged into viral vectors and... L, Campbell RE. Structure-and mechanism-guided design of single fluorescent protein-based biosensors....

...FLP and Cre for additional control   Figure 2: Expression of a morphological marker (eYFP) and synaptic...Sternson. 2008. “A FLEX Switch Targets Channelrhodopsin-2 to Multiple Cell Types for Imaging and Long-Range ...PMID: 18614669. PubMed Central PMCID: PMC2593125. 2. Buchholz, F, P O Angrand, and A. Francis Stewart....The FLP Recombinase of the Saccharomyces Cerevisiae 2 Micron Plasmid Attaches Covalently to DNA via a Phosphotyrosyl... this re-inversion issue. It became possible to design FLEx vectors (Flip-excision vectors) that only ...

...pH visible at the sample interface upon thaw (Fig. 2). Before thawing, all the samples were at the control...showed that AAV infectivity was not altered by a short 2-hour exposure to pH as low as 2.5 (Potter et al., ...Methods. 2013 Apr;10(4):278-9. PubMed PMID: 23538862. 2. Potter M, Lins B, Mietzsch M, Heilbronn R, Van Vliet...shipment processes. After reading a publication describing how short term storage on dry ice can shift sample...

...component can be designed with an overhang specific to the type of module it is (Pol 2 protomer, Cas protein... of components, such as your CRISPR enzyme or Pol 2 promoter. The options available in each class, or ... (KO, CRISPRa, CRISPRi, editing)   Figure 2: Selecting module fragments on the Fragmid website...list of Fragmid destination vectors currently available. Table 1: Fragmid destination vectors Organism...website, which breaks the design process down into four easy steps, provides the real value for the user...potential of them not working, or spend a few weeks designing and cloning the vectors you actually want. But... Fragmid is a modular vector assembly process designed for a quick, plug-and-play approach to cloning...

...neoblast cells and the gene Prohormone convertase 2 (pc2) labels the central nervous system. Evaluating... characterized in an unrelated worm (C. elegans) [2]. Delivering dsRNA matching a specific mRNA sequence...223). Link: http://abt.ucpress.edu/content/79/3/208 2. Fire, S. Xu, M.K. Montgomery, S.A. Kostas, S.E. Driver...A recent publication [1] from the Sánchez lab describes the plasmids and protocols required for both WISH...companion resource called “cutting class” which provides more detailed materials and protocol resources...synthetically made RNA (riboprobes) with modified nucleotides (for example, Digoxigenin-labelled Uracil) that... molecular cloning to insert the cDNA into the desired plasmid backbone. The Sánchez lab plasmids allow...

...retinal cell-type specific genes from mice. Strategy #2 (ProB): Create promoters using phylogenetically conserved...would express Cre-mCherry and GFP.   Figure 2: Synthetic promoters can be paired for targeted expression...1345-1356. https://doi.org/10.1038/s41593-019-0431-2  Neff, E.P. An AAV library for retinal cell expression...expression in the retina.  Four approaches for designing synthetic promoters  To build a library of synthetic...synthetic promoters, the Roska lab used four different design strategies: Strategy #1 (ProA): Create promoters...promoters with the help of epigenetic analysis and designed using stretches of DNA that were transcriptionally...mice. Figure 1: The four strategies used to design synthetic promoters. Legend: TSS: transcription...

... fluorophore excitation by absorption of a photon 2. Energy transfer from the excited donor directly to...PMID: 24194740. PubMed Central PMCID: PMC3810607. 2. Bajar, Bryce T., et al. "A Guide to Fluorescent Protein...PubMed PMID: 10500161. PubMed Central PMCID: PMC18018. 2. Miyawaki, Atsushi, et al. "Fluorescent indicators...Resonance Energy Transfer (FRET) was originally described by Theodor Förster in 1948 as a variation of the... observed light emission by fluorescence. The widespread use of FRET with fluorescent molecules, including...FRET probe, try searching PubMed for articles describing the FRET tool that you are looking for and check...created the sensor that you need. FRET biosensors designed to measure specific small biomolecules or gene...

... shown in red in the spreadsheet (Figure 2) and are described in Table 1. Some cells have drop-down options...our deposit spreadsheet to complete Step 1. Steps 2 and 3 are usually very easy — our tech transfer team...Filling out and sending the deposit spreadsheet Figure 2: Example of the deposit spreadsheet showing the first...spreadsheet Data Field Description Plasmid Name Use a descriptive plasmid name and match the...lab. Plasmid Type Choose from: Encodes one insert, encodes gRNA/shRNA, or empty backbone. Purpose...does this plasmid do?” in <200 characters. A descriptive purpose focuses on the practical application ... Primary Vector Type The vector type should describe how your plasmid is intended to be used. Choose...