...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...

...super-resolution or single-molecule techniques.   Figure 2: Two-color super-resolution imaging of ER and mitochondria...stimulated emission depletion (STED) nanoscopy. Scale bar, 2 μm. Inset shows comparison with standard confocal ...enables applications like multi-color imaging (Figure 2). If you’re working with purified proteins in vitro...Labeling in Living Cells. Chemistry & Biology, 15(2), 128–136. https://doi.org/10.1016/j.chembiol.2008.01.007...conjugates: A flexible chemical tag. Nature Methods, 2(4), Article 4. https://doi.org/10.1038/nmeth749 Strebinger...Cell type-specific delivery by modular envelope design. Nature Communications, 14(1), Article 1. https...

...incubation at 37 °C. Wash the membrane for 5–10 minutes 2–3x in fresh wash buffer such as PBS or TBST. Stringent...Purpose Tris HCl 62.5 mM Buffer SDS 2% (w/v) Detergent Β-mercaptoethanol (βME) 100...minutes at 50 °C. Wash the membrane for 5–10 minutes 2–6x in fresh wash buffer such as PBS or TBST. Note ...v) HCl Adjust to pH 2.0–2.2 For the mildest stripping solution, omit the Tween 20. Once your...

...PCR is designing primers. There are 3 strategies for primer design: 1) insert-specific primers, 2) backbone-specific... to colony PCR are: 1) design primers to detect the presence of your insert; 2) set up a standard PCR ...negative clone (-) will not. Backbone-specific primers (2) give larger sized products for positive clones (+... second option is to design backbone-specific primers. These primers are designed to anneal to sites that...things to consider when performing colony PCR.   Designing colony PCR primers The first and perhaps most ...-specific primers: Insert-specific primers are designed to anneal to an insert-specific sequence. This...backbone but that contain different inserts. When you design primers to anneal outside the cloning site, it ...

...Sullender M, Ebert BL, Xavier RJ, Root DE (2014) Rational design of highly active sgRNAs for CRISPR-Cas9...posts: Part 1: Genetically Engineered Mice and Part 2: Breeding and Crossing Mice. My first CRISPR mouse...is less predictable. In our best case, we saw that 2 out of 3 mice carried a 5 kb insertion in the ROSA...LO, Morgan JL, Juan WC, Roca X, Ong ST, Bergstrom DE (2018) Viable Mice with Extensive Gene Humanization... mouse genome is made up of 3 x 109 nucleotides (nt), and encodes 23,000 or so genes. It would be great...guide you in gRNA design. We have been using Benchling, but there are many other gRNA design tools available...for cells that had successfully incorporated the desired mutation, and then derive mice from these engineered...

...or cloning, as digestion of the destination vector with SapI yields 2 sites- the first site accepts the...marker, and connector to be released (Fig. 1, Step 2-3). Since donor plasmids of the same type will produce... a gRNA vector that targets the gene of interest, 2) a plasmid to specify the reading frame of the insertion...SapTrap, the user first designs either oligos or synthetic DNA for the desired gRNA target sequence, as...specified by a guide RNA (gRNA) sequence. Users can design gRNAs to induce the break at a specific genomic...accepts the homology arm repair template. SapTrap includes a prebuilt donor plasmid library containing several... and a Cas9 expression plasmid will insert the desired genetic tag and marker sequence into the targeted...

...undergraduate students (1, 2), but educators face significant challenges in designing suitable projects. In... by western blotting in other experiments (Figure 2).   Complementation reveals conservation of function...Biology Education: A Call to Action, Washington, DC. 2. President's Council of Advisors on Science and Technology...Over Time project The Pathways over Time project provides a framework for these undergraduate research questions...

... is also gaining a lot of new traction lately (1, 2), depends on knowledge of the structure(s) a given...per plate) across 20 fresh selective plates. Step 2: Express random phage DNA fragments in E. coli Now...microbiology 23.4 (2015): 185-191. PubMed PMID: 25708933. 2. Cooper, Callum J., Mohammadali Khan Mirzaei, and ... biology 34 (2015): 69-77. PubMed PMID: 26275959. 2. Weigele, Peter, and Elisabeth A. Raleigh. "Biosynthesis...identify possible gene function. In this post, I will describe a useful and cost-effective method that our lab...and probe with live cells The following is a description of a technique published by Simpson, Sacher, ...ends) and ligate into a bluntly digested vector designed for protein overexpression in E. coli, such as...

...the system outlined in Schmid-Burgk, J.L., et al (2). In this post, I’ll cover two great applications ...molecularly evolved from mNeptune2 through 33 mutations and 2 deletions. CyOFP1 has a higher quantum yield, brightness...luciferin 24 hours later demonstrated a more than 2-fold brighter signal from Antares as compared to Fluc...PMID: 22894855. PubMed Central PMCID: PMC3501149. 2. Schmid, J.L., et al. (2016) CRISPaint allows modular...above 600 nm. LumiFluors Schaub, F.X., et al. (3) designed two imaging probes. One consists of NLuc fused...Reporters. The LumiFluor reporter constructs were designed to be packaged into a retrovirus for infection...BRET probe for imaging. These authors set out to design a better fluorescent protein that could be co-imaged...

...Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system." Cell (2015). PubMed PMID: 26422227... Find plasmids from this publication at Addgene. 2. Kleinstiver, Benjamin P., et al. "Genome-wide specificities...Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System. Cell. 2015 Sep 23. pii: S0092-8674...for Cas9 (42 nt vs 100 nt.) However, the newly described non-CRISPR protein NgAgo is smaller than any of... of these options (2.7 kb with 24 nt guides), so it has the potential to eclipse any of the CRISPR nucleases...to examine Cpf1 off-target editing. First, they designed gRNAs with single and double mismatches throughout... endjoining (NHEJ). Cas9-mediated NHEJ usually destroys the PAM site due its proximity to the cleavage...

... characteristics of select AAV serotypes in Table 2 from the paper. Delivery of AAV into the brain can...routes for neuroscience see Table 1 from the paper. 2. Manipulate your cell of interest: Cell-type specific...are the three main takeaways from the paper for designing experiments with AAV: 1. Getting your viral vector...AAV from our viral service can share experimental design and data. With these different approaches to open...

...groups, such as their vaccine production, SARS-CoV-2 research, or adenovirus packaging cell lines dataset...-Line Knowledge Resource. J Biomol Tech. 2018; 29(2): 25-38....database describing nearly 145,000 cell lines. Cellosaurus Cellosaurus seeks to identify and describe all ...authors of the paper in which the cell line was described. Using Cellosaurus Cellosaurus is searchable by...Cellosaurus received a report that HT-55, which was described as a colon cancer cell line, is actually a rectal...

...the result in Figure 1 could look like this: Figure 2: Expression of Plasmid B (as internal control) This...down expression of Gene X but, as shown in Figure 2, this was likely due to the original transfection ...PMID: 20074371. PubMed Central PMCID: PMC2817684. 2. Replicates and repeats—what is the difference and...and should be designed accordingly. If you are trying activate a gene, you should design a control that...carried out by scientists every day. Although the designs and outcomes may vary, one thing should be present...these control plasmids are critical for the correct design of the experiment.  The Experiment: Knockdown the...control plasmids Part of planning your experiment includes determining what factors need to be controlled...

...scientists have 1) the courage to make mistakes and 2) the integrity to recognize these mistakes quickly...policies that “muzzle” government scientists to the de-funding of crucial environmental research programs...She studies beta-cell function and failure in Type 2 Diabetes. Birmingham, AL, USA 33.5207°N 86.8025°W ...available to people who want to join the dialog and 2) remember that science should not be partisan: everyone...and minority communities, where these issues hit hardest. According to the Guttmacher Institute, most women...was supremely proud of Nashville that morning. Despite the rain, about 4,000 supporters of science showed... an agenda that halts current data collection, destroys existing data, and guts future funding.I marched...

...inhibit editing (Anzalone et al., 2019). Figure 2: Starting lengths for optimizing your pegRNA primer...great tips to guide your experimental design, especially the design of the RNA sequences you’ll use in prime...principles pegRNA design If you’re just starting out with prime editing, you’ll need to design and optimize...reverse transcriptase (RT) template including the desired edit. Figure 1: Prime Editing schematic.... be edited and the sequence of the edit, so its design changes for every new prime editing application...transcriptase template, starting with about 10–16 nucleotides. For longer templates, it is even more important...and PE5b: In this approach, the nicking sgRNA is designed to bind only after the edit is installed. This...

...indicating primer dimers. (c) A double peak indicating 2 amplicons. Image credits (respectively): Zuzanna K...details for reproducibility’s sake!   Fig. 2: Setting up triplicates in multiple configurations...used method is the Standard Curve method. See Table 2, (adapted from Wong, 2018) for a comparison of different...Statistically defined log-linear phase   Table 2: A comparison of different qPCR analysis methods. ... for RNA quantitation. Biotechniques. 2000 Aug;29(2):332-7. doi: 10.2144/00292rv02. PMID: 10948434.Taylor...from Wong, 2018   Primers Design of primers and probes Primer design, of course, could easily be its...useful primer design resources in the References and Resource section. Validation Once designed, your primer...

...handled at BSL-1, while lentivirus requires a BSL-2 setup. This requires a less involved setup when working...infection rates in your experiment. Figure 2: Visual representation of common AAV serotypes used..., 10(1). https://doi.org/10.1038/s41467-019-12449-2 Pupo, A., Fernández, A., Low, S. H., François, A.,... This vastly increases the possibilities when designing screens. Each serotype has its own tropism, or...experiment. Additional considerations One of the downsides to using AAV is the limited packaging capacity...Adames, A. C., Saur, D., & Rad, R. (2022). Tutorial: design and execution of CRISPR in vivo screens. Nature...

...(you generally don’t want more tag than protein), 2-3 tags is increasingly common. Tandem affinity purification...TEV cleavage site (more on that in a moment), and 2 ProtA IgG-binding domains. TAP has since come to encompass...found in this paper (available at Addgene).   Table 2: Protease recognition sites commonly used with tags... Protein tags are usually smallish peptides incorporated into a translated protein. As depicted in the...depending on their protein of interest. After several decades of trying to address these challenges, researchers...examples. The tables list more common tags than are described in the post, but have been categorized to help...

...Epub 2016 May 2. PubMed PMID: 27136078. Find plasmids from this publication at Addgene. 2. Swarts, Daan...DNA guides NgAgo uses 5’ phosphorylated DNA guides (so called “gDNAs”) rather than the RNA guides employed...24 base DNA guides may be more user-friendly, since they can be ordered as oligonucleotides and should ...gDNA guides will also need to be transfected into the cells of interest. In contrast, RNA guides must ...important role in RNA interference, binding RNA guides to cleave foreign RNA. Argonaute proteins are also...cleave DNA in vitro, directed by phosphorylated DNA guides. Unfortunately, Swarts et al. were unable to show...NgAgo preferred certain guide sequences - using 5 guides/gene for 10 genes, they did not observe differences...

...base pair to another base pair at a specific site. 2. Temporal, spatial, or conditional control of CRISPR...difficult-to-engineer Sulfolobus islandicus rod-shaped virus 2 (Mayo-Muñoz et al., 2018). Only viral particles that...Editing of Sulfolobus islandicus Rod-Shaped Virus 2. Viruses 10:695 . https://doi.org/10.3390/v10120695.../35++ Adenovirus Vector Expressing Anti-CRISPR Peptides Decreases CRISPR/Cas9 Toxicity in Human Hematopoietic...

...3 plates at different densities for selection). A 2% agarose solution is then melted (and thereby sterilized...96-well assay plate. We generally freeze at least 2 ampules each of 4 clones with different levels of ...of agarose, which is then overlaid with the firmer 2% agarose gel solution discussed above for picking....investigators to identify specific clones with desirable properties, either initially, or using secondary...