We narrowed to 718 results for: des.1

...mutation (usually 1-3 mismatched bases) is sufficient for your primers to successfully anneal to the plasmid...researcher at Cornell University.       References 1. Breslauer, Kenneth J., et al. "Predicting DNA duplex...for the desired modification. Finally, the positive clones are sequenced to confirm the desired modification...Primer Design As a rule of thumb, 11 bp of complementary sequence on either side of the desired mutation...introduced to plasmids using primers (with the desired mutation) in a PCR protocol that amplifies the ...mutated clones. Forward and reverse primers are designed to be complementary, but each primer may extend...Polymerase To ensure that you don’t introduce undesired mutations through the PCR process, you need a ...

...folding, and deal with sub-optimal codon usage (Table 1) A few mutations are common to all or most expression...curated list of Bacterial Expression Systems Table 1: E. coli expression strains  Note: All strains are...and some of the most popular commercial strains designed to avoid them. Why do I need an expression strain...protease: Strains where this is completely deleted (designated lon or Δlon) similary reduce proteolysis of the...which can prevent the culture from reaching the desired density within a reasonable time-frame. For these...and pLysE—the difference being that the latter provides tighter control of basal expression. What if I...don't see protein overexpression? The strains described above should generate sufficient expression levels...

...Time for the #BioSci3319 #StreakPlateChallenge part 1. Lab B needs a redo :-( @SandTBioSci #CompetitiveStreaking...the Streak Plate Challenge, Westenberg’s course includes Winogradsky columns and characterizing the microbes...

...know that most cephalopods die after they lay their 1 clutch of eggs? Propagation is thus quite difficult...organism blog posts: Five Popular Model Organisms, Part 1 Five Popular Model Organisms, Part 2 ...promotes scientific sharing The EDGE Program is designed to expand the number of systems that can be studied...genotype to phenotype studies New organismal systems described at this meeting were extraordinarily diverse. ...generating interesting drug-like compounds especially cardenolides which have been used to treat heart disease....disease. Caterpillars that eat milkweed generate cardenolides that tastes yucky (and is poisonous) for bird ...control who a female wants to mate with and if she decides to raise their offspring or leave them to their...

... new preps might enhance your research!   Table 1: New ready-to-use viral vectors in the Addgene repository...community and support a wide range of experimental designs. Here are nine great additions, including our first...

...an antibody performed in an experiment.   Figure 1: The front page of the Addgene Data Hub.   Antibody...antibody worked under the conditions described above, and a short description of how the antibody performed....the submitted application, along with a brief description of the experiment. If that data was included ...ELISA — but in all cases, it contains a detailed description of the experimental conditions.     Figure 4...The submission process is easy, with simple, descriptive fields, drop-down boxes when appropriate, and...

...sample’s absorption at 260 nm (A260). Figure 1: Characteristic absorption spectra for pure DNA and.... Issue 15/10. March 2010. https://www.qiagen.com/de/~/media/1ea8aec3bfa24543a28fcaea25986514.ashx. Koetsier...

...activity to a transcriptional activation domain (Fig 1). Two ways to use CRISPRa for your research CRISPRa... Stucka R, Hauber J, Lange UC (2016) Targeted HIV-1 Latency Reversal Using CRISPR/Cas9-Derived Transcriptional...whichever guides are closest to the transcription start site (TSS). We recommend targeting the guides to a ... you should also be ready to test three or four guides directed towards that gene as there can be a large...transcription. That being said, we generally pick guides by putting the promoter of the gene into a gRNA...

...straight to your materials of interest!   Figure 1: The new search bar.   Pro tip! You can use a manuscript...and the Addgene Help Center. A big part of this redesign was pulling all of our science options into a ...Addgene’s website easier to navigate, thanks to design choices informed by Web Content Accessibility Guidelines...word for it — check out all the new features and designs addgene.org! And when you’re finished, feel free...

...forward to another great year of sharing!   References 1. Shalem, Ophir, et al. "Genome-scale CRISPR-Cas9 knockout...Feng Zhang’s lab at the Broad Institute and was designed and cloned by Neville Sanjana. This fantastic ...your gRNA of interest to mammalian cells. It was designed to have increased lentiviral titer as compared...sampling of the research it has already been used for includes work probing mTOR signaling in cancer cells, deciphering...

...Figure 1). Clustered events are called a population.                             Figure 1: While...series of markers are set up sequentially, from broadest to most specific marker, and are represented as...are, try drawing a quadrant over the plot that divides it into four, using the location of the cell populations...Gating and plot orders  As the gates progress from broadest markers to most specific, the first step is to...

...breaks (DSBs) or donor DNA templates.  Figure 1: An overview of prime editing. Created with BioRender.com...demonstrated ~20-50% efficiency in HEK293T cells with 1-10% indels. Since then, further innovations have continued...Engineering. https://doi.org/10.1038/s41551-024-01227-1 Yan, J., Oyler-Castrillo, P., Ravisankar, P., Ward...site by the pegRNA — a guide RNA that also encodes the desired edit and homology to the genomic DNA locus...both specifies the target site and contains the desired edit (or edits) engages the prime editor protein...and the reverse transcriptase domain copies the desired edit, directly polymerizing new DNA onto the nicked...edit within what Fyodor Urnov refers to as “PAM deserts” in the genome. More versatile and precise than...

...older epitope tags - only time will tell! References 1. Georgieva, Maya V., et al. "Inntags: small self-structured...First described in the 1980s, protein tags are now one of the most useful items in a scientist’s toolbox...IT10 expressed well in cells and did not form undesirable aggregates. Using budding yeast as a model system...

...vectors, and many more, in the table below.    Table 1: New ready-to-use viral vectors in the Addgene repository...pAAV-CAG-DIO-NLS-mRuby3-IRES-eGtACR1-ST AAV9 Optogenetics Adesnik New viral vector pAAV-nEF-Con/Foff DREADD Gi-...

...$47,476 and the date for implementation was set for Dec 1 2016. In order to be exempted from the need to pay... these institutions (populations potentially from 1 to 5761, according to the NSF data) and therefore ...around $45,000, but as I’ve discussed elsewhere (slides here) there are many postdocs paid at much lower...expected for postdocs in many cases should be as described in the notice of the new NRSA levels and in the...

...making it easier for us to do our work.” References 1. Athanasios Lampropoulos et al. "GreenGate - A Novel...colleagues describe in the PLoS ONE paper that introduced the kit in December, GreenGate includes the six... bring this into your target organism. We have designed a Golden Gate system based on the daily needs ...and cut another,” Lohmann said. “It allows us to design modules that always have the same recognition site...produced will have different overhangs, they can be designed such that they go back together only one way and...

...together along with the Gibson assembly master mix for 1 hour, and then transform into competent cells. The...you should consider giving it a try! References: 1. Gibson DG, Young L, Chuang RY, Venter JC, Hutchison... friends on the Cambridge 2010 iGEM team that describes the basics of Gibson assembly as a parody of “...assembly The Gibson assembly technique was first described by Dr. Daniel Gibson and colleagues at the J. ... Both Snapgene and NEB have tools that help you design primers for PCR amplification of fragments to incorporate...demonstration of how to use Snapgene’s program to design primers for Gibson Assembly. For a simple example...without scars as depicted below. First, you need to design primers to amplify the two fragments while also...

...improved sensors for these and other targets. Figure 1: MTRIA biosensors are generated by inserting a circularly-permuted...fluorescent biosensors. Nat Commun. 2022 May 25;13(1):2919. doi: 10.1038/s41467-022-30685-x. Feel the ...Addgene’s catalog is a new class of sensors for neuropeptides and neurotransmitters. Motivated to develop ...GPCRs recognizing various neurotransmitters, neuropeptides and other ligands - almost A to Z! - from ATP...a new preprint, Jared Toettcher and colleagues describe pYtags, a modular system for monitoring specific... tool for monitoring RTK signaling.  Figure 4: Design of pYtag fluorescent biosensors for monitoring ...iGECInano plasmids here!   Matlashov ME, et al. Design and Initial Characterization of a Small Near-Infrared...

...at a low angle (FSC) or large angle (SSC) (Figure 1). FSC values depend on the cell's size, while SSC ... or ask experienced colleagues.   Figure 1: The laser pulse illuminates the cell and is scattered... your cells based on their size, the cytometer provides you with two measurements: forward scatter (FSC...SSC-H, or FSC-W/SSC-W, respectively. H, A, and W describe the shape of a histogram, which graphically represents... of the cell’s illumination (see Figure 2). H describes the maximum signal strength, while W results from...photocurrent vs. time. The resulting histograms is described through maximum current (H), the time the cell...depends on cell type and laser voltage. The voltage describes an electric potential that can be applied to the...

...compare the experimental group.     Figure 1: A, Most biological experiments will benefit from ... Histochemistry & Cytochemistry, vol. 59, no. 1, SAGE Publications, Jan. 2011, pp. 6–12. Crossref,...controls and be able to use this information to design thoughtfully and thoroughly controlled experiments...is generally a sample or group that will have a desired response. For example, if you are studying the ...sample, the antibody binds specifically to its desired target (purple) and nonspecifically through the...The Preimmune serum control will not bind to the desired target but will bind nonspecifically through the...the Fc, allowing you to detect this undesirable interaction. When non-specific interactions are suspected...

...diseases. References 1. Harrington, Lucas B., et al. "Programmed DNA destruction by miniature CRISPR-Cas14... cas1, cas2, cas4, and a new gene cas14. cas14 encodes for a small Cas protein (40-70 kDa), which is half...

...constructs to the kit in the next two years. References 1. Martz, Colin A., et al. "Systematic identification...so he assembled a plasmid kit designed to do exactly that. As described in the journal Science Signaling...When cancers are treated with drugs designed to hit them right where it hurts, the effects are often ...mutant complementary DNAs (cDNAs) representing core nodes that, when over-expressed, constitutively activated...activators for more cancer-related pathways and more nodes within those pathways. With support from the National...

...Lab's Nature Protocols Publication describing them here. References 1.Ran, F. Ann, et al. "Genome engineering... and the Puromycin resistance gene as separate peptides. PX459 This is the vector backbone. This particular...Protocols Paper for more information). V2.0 This designation separates pSpCas9(BB)-2A-Puro (PX459) V2.0 from...Guide Pages Browse Our CRISPR Collection Find gRNA Design Tools ...

... remains fluorescent in acidic organelles (Figure 1), showing that its acid tolerance is maintained in...follow him on twitter: @joachimgoedhart.   References 1. Zacharias, David A., et al. "Partitioning of lipid-modified...cofactor binding proteins as described above. The smallest tags are peptides that are engineered to bind...homology domains can be used to detect specific phosphoinositides (Varnai and Balla, 2008) (Figure 2). RNA production...Péter, and Tamas Balla. "Live cell imaging of phosphoinositides with expressed inositide binding protein domains...

...single neurons in the living brain   Figure 1: Plasmid mix to label neuronal morphology (eYFP) and...genOway press release for more information. References 1. Atasoy, D., Y. Aponte, H. H. Su, and S. M. Sternson... this re-inversion issue. It became possible to design FLEx vectors (Flip-excision vectors) that only ...

...downstream experiments. You only successfully generate 1 null clone and proceed with it. In this case, the ...there are 10 putative off-target locations you can 1) determine if editing at those sites would confound..., it’s essential to understand them in order to design the most specific assays possible. In this blog...-target effects should impact your experimental design differently. Yes, it is always better to reduce...introducing a specified edit). However, if your design does have wiggle room, you can minimize off targets...

...in the crystal jellyfish Aequorea victoria (Figure 1) in molecular detail. On the underside of the “umbrella...characteristic radiance for some time – there are records describing them dating back to the first century A.D. – ...mechanistic understanding of GFP’s function. His lab described the protein’s structure and also discovered many...

...communication check out these SciComm blog posts. References 1. Bourdeau, Raymond W., et al. "Acoustic reporter genes...is absolutely fascinating to see the living art designed by creative minds with a (I assume) very steady...Our Microbiology Resource Page CRISPR plasmids designed for use in bacteria Deposit Your Microbiology ...

...to the interview with Science in the News Chapter 1: Introduction - Vini and Amy tell us a little about... discusses some of the programs SITN runs and describes some of the more popular topics from the Science... fields as well as students from the ed school, design school, and Kennedy school to discuss the impacts...impacts of science in a social context. Episodes are driven by narrative and story telling. Chapter 5: The...The SITN Blog - Vini and Amy describe how the SITN blog is a source of scientific information for the ...underwater? Chapter 8: Work-Life Balance - Vini describes how she balances working in the lab and working...

...may contribute to more in the future. Figure 1: A timeline of CRISPR base editing advances that led...Lander, E. S. (2016). The Heroes of CRISPR. Cell, 164(1), 18–28. https://doi.org/10.1016/j.cell.2015.12.041...performed best when paired with the V106W mutation (described earlier) to minimize off-target editing. Base ...final base editor that was administered to KJ was designated NGC-ABE8e-V106W. The name is hardly flashy, but...

...Cloning grade DNA Transfection grade DNA Figure 1: Transformation introduces transformation grade plasmids...other uses.   Cloning grade DNA Molecular cloning includes a set of techniques that are used to insert recombinant...

... successful after their training.   Figure 1: Professional Development Hub Workshop Attendees and...job of providing skills training for scientists destined to continue in academia. It seems it’s largely...

... with DNA sequencing troubleshooting and analysis 1. You can use any of the following programs to view...are usually unresolved and small, so I suggest designing your primer at least 50bp upstream of the sequence...

...is here to help. Contact us at [email protected] or +1 617-225-9000, or check out our MTA reference pages...University Technology Managers (AUTM). This MTA provides many benefits for both the providing and receiving...

...normal size.      References: 1. Doench, John G., et al. "Optimized sgRNA design to maximize activity and ... producing a wall of results that can only be described as bigly. But such a project should not be undertaken...Library Screening Why the kinome? While properly designed perturbations – in the case of screens using CRISPR...with CRISPR/Cas9 Get John Doench's Tips on gRNA Design Check Out News Ways to Use CRISPR for Single Base...

...organized and boost your productivity in 2014. Tip 1: Use color coding, labels, and post-its to make finding...lab earlier, to read that stack of papers on your desk, and to tackle some new experiments before the next...gathering that pile of lab notebooks and binder on your desk. Arrange them in chronological order (or by project...labels. Color code your Post-its and write brief descriptions on each. Place the notes sticking up out of ...colleagues. Start small by identifying the protocols, guides, or references that your labmates ask to borrow...

...science.aaq0180  Fine EJ, Appleton CM, White DE, Brown MT, Deshmukh H, Kemp ML, Bao G (2015) Trans-spliced ...indel formation and phenotypic changes as early as 1 week post-injection. Livers from these mice were histologically...as potent and specific? The two AAV strategies described above showed successful target modification, indicating...from Streptococcus thermophilus and a rationally-designed truncated Cas9. Unfortunately, certain drawbacks... approximately 1350 amino acids in size, which includes SpCas9, and shortern orthologs approximately 1000...Split inteins are a pair of naturally occuring polypeptides that, when at the ends of two proteins, mediates... proof-of-concept split intein SpCas9 which had modest editing rates in HEK-293T cells when compared to...

...target cell line or organism (Presented in figure 1). More information on Agrobacterium-mediated transformation...mediated cloning of your gRNA of interest. Figure 1: Simplified schematic of agrobacterium-mediated transformation... While it is certainly possible to select for desirable traits using traditional plant breeding approaches...modification of known or suspected genes that regulate a desired phenotype. In fact, CRISPR has already been used...wheat, mushroom, and rice (Khatodia et al., 2016). Despite the almost universal functionality of the CRISPR...genes in plants using the same general experimental design principles developed in other model organisms (...nanostructures (Zhang et al., 2019) or cell-penetrating peptides (Santana et al., 2020) and plants viruses (barley...

...individually or as a complete kit from Addgene. References 1. Goulas, Theodoros, et al. "The pCri system: a vector...subtilis and Pichia pastoris pCri also includes plasmids designed for protein expression in systems other...construct, most often in special E. coli strains designed for protein expression. Cultures are then lysed...plasmids are divided into two sets, each of which includes plasmids with different varieties of tags, both...other eukaryotes better than E. coli. Plasmids designed for these systems may include a signal peptide...can improve disulfide bond formation. pCri also includes a special feature for difficult-to-purify membrane...membrane proteins. pCri vector pCri-13a, designed for use in B. subtilis, contains the MISTIC (membrane-...

...reconstitutes a fully functional luciferase enzyme (Figure 1). Addition of substrate generates a proportionate ... before creating knock-ins.  Figure 1: Schematic of HiBiT tagging technology.   The bioluminescence...most clinically-relevant proteins are also the hardest to study. One common strategy is protein overexpression...compatible with both strong and weak promoters, and even modest endogenous pools generate high signal-to-background...introduce mutations. This vector-based use of HiBiT provides a means to quickly answer research questions before...the code of 'undruggable' targets Researchers can design drugs to bind to well-defined ligand-binding pockets...mechanistic drug responses (Kamio et al., 2025). They designed HiBiT-tagged TP53 constructs, one with an R175H...

... and deep-brain fiber photometry.  Figure 1: Characterization of iGluSnFR4 in mice. A) Scheme ...CycT1 or PolII-CTD antibodies and unlabeled MS in DLD-1 cells (colorectal cancer). Image courtesy of Brianna...Glutamate-Sensing Fluorescent Reporter (iGluSnFR) designed and improved by the Loren Looger and Kaspar Podgorski...Aggarwal et al., 2025).In this latest work, the team designed and screened a large library of iGluSnFR3 mutants... HA-, and EGFP-expressing lentiviral backbones designed right here at Addgene. Here's what makes these... stable cell line creation a breeze. Modular by design: Key elements (promoter, tag, polyA site) are flanked... plasmids for flexibility and speed, so you can design, test, and innovate faster than ever. Plus, we'...

...deletions, and all possible base-to-base changes.   1. Plasmids 101: Transformation, Transduction, Bacterial...on the Addgene blog and have created and updated guides on various topics in biology over the past year...containing these fluorescent proteins at Addgene. 7. Designing Your Chalk Talk for the Academic Job Interview...

...products by their environmental impact on a scale of 1-10: the lower the score, the lower the environmental...collect in those red biohazard bags, typically is destined for a landfill. However, Triumvirate Environmental...Then, they use this material to produce various grades of plastic lumber and distribute it to industrial...

...resume that will give you a chance at an interview:  1) Have an excellent resume ready to be submitted The...experience. Most Addgene PhD level job descriptions clearly describe that the roles do not involve bench ...submission of a job application. 3) Read job descriptions carefully Be open minded and be bold. You don...qualifications to apply for a position.  A job description is usually a wish list and the hiring manager... way to a hiring manager. Learn to confidently describe your strengths in your resume and don't be shy...

...to western blotting  In a western, proteins are: (1) separated by size, (2) transferred to a membrane,...  (3) detected using antibodies.   Figure 1: Overview of the Western blot process. The sample,...protein analyses using ELISA. Peptides 72:4–15 . https://doi.org/10.1016/j.peptides.2015.04.012 Burnette WN ... Figure 2: The western blot dry apparatus includes a stack of filter paper, a copper anode/iblot ...blocking buffers, and incubation times. If the vendor includes publications that cited the antibody, consider...abundance In addition, the thickness of the band provides information about the relative abundance of the...

...five methods for extracting DNA without a kit too. 1. Early Career Researcher Toolbox: Free Online Molecular...databases, DNA sequence manipulation, and primer design. Best wishes for the new year and your research...

...2013 edition. 10 new developments in open science 1)      In February, the Office of Science and Technology...     Science’s John Bohannon published an article describing a sting operation in which he submitted a phony...

...furimazine by Promega’s Chemistry group is a good story (1). From a deep sea shrimp to a compact powerhouse of...cellular analysis products at Promega.     References 1. Hall, M.P., et al. (2012) Engineered luciferase reporter...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...acceptor, the more powerful the FRET signal. In well-designed FRET-based biosensors, the intensity of the FRET...

...relations, advocacy, and writing legislation (see Figure 1 for career map). The beautiful part of science policy...about science policy as a profession. Figure 1: Science policy can lead you to many paths! For a ...uncertainty of it all. Though I had spent the first few decades of my life following a very straightforward conveyor... harassment in academia or a large company who decides how they are going pursue regulatory approval for...their Local Engagement Playbook for step-by-step guides on how to engage in your local community.  Read...comprehensive resource developed by Dr. Kelly Singel which provides an overview of all available science policy fellowships...

..., or CaURA3 selection marker. Lee et al.(1) assessed many of these fluorescent tags in S. cerevisiae, ...protein products is desired, as multiple selection markers can ensure that all desired tags have been integrated...integrated. Simply design your amplification primers with the desired targeting homology—in frame, of ...These tagging vectors are based on previously described pFA6a-link vectors and include a Kan, SpHIS5, ...former lab at UNC Chapel Hill. Bähler et al.(2) describe a modular collection of plasmids for a wide variety...3xHA, 13xMyc, GST, or GFP). Longtine et al.(3) describe a complimentary set of plasmids for use in S. ...