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We narrowed to 1,025 results for: URE

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  1. Choosing a Good Mentor for Scientists

    Type
    Blog Post
    ...post about workplace bullying featured on the always interesting Tenure, She Wrote blog. Unfortunately...little pre-work most PhD students and postdocs do to ensure the advisors they choose will be ones that help...choose a field that you may want to work on in your future career, so the research topic will be more important...scientists to change labs – do as much as you can to ensure a good fit in advance. When a lab is interviewing...interviewing the lab to find a good “fit” and to ensure the PI will be a good role model and teacher. Do...creates a level of familiarity and confidentiality to ensure more honest answers. Ask people who work in the... to speak to them more than once to get a full picture and listen for hints at the same concerns from ...

  2. Proximity Labeling: A Powerful Tool for Protein Complex Purification and Proteomic Mapping

    Type
    Blog Post
    ...methods termed proximity-based labeling to better capture these interactions in a vast array of biological... of biotin for avidin, proximity labelling can capture weak/transient protein interactions as well as ...exposed lysines on neighboring proteins. Thus to capture protein complexes, scientists can simply express...After a few hours biotinylated proteins can be captured on streptavidin affinity beads or matrices. The...labeling in living cells and organisms with TurboID." Nature biotechnology 36.9 (2018): 880. PubMed PMID: 30125270...for electron microscopy and proximity labeling." Nature methods 12.1 (2015): 51. PubMed PMID: 25419960....genetically encoded reporter for electron microscopy." Nature biotechnology 30.11 (2012): 1143. PubMed PMID: ...

  3. Evolution of Brainbow: Using Cre-lox for Multicolor Labeling of Neurons

    Type
    Blog Post
    ...necessitating the development of a new technique. In a structure as tightly packed and interwoven as the brain,...previously visible with Brainbow-1 and -2. The general structure of Brainbow-1.0 is retained in Brainbow-3.0, but..., but can be detected via immunostaining. This feature facilitates screening of Cre-negative Brainbow ... H, Draft RW, Bennis RA, Sanes JR, Lichtman JW. Nature. 2007 Nov 1;450(7166):56-62. PubMed. A technicolour...the connectome. Lichtman JW, Livet J, Sanes JR. Nature Reviews Neuroscience. 2008 Jun;9(6):417-22. doi... P, McKellar CE, Hall D, Looger LL, Simpson JH. Nature Methods. 2011 Mar;8(3):253-9. doi: 10.1038/nmeth..., Alexandre C, Bell DM, Dickson BJ, Salecker I. Nature Methods. 2011 Mar;8(3):260-6. doi: 10.1038/nmeth...

  4. Antibodies 101: Avoiding the Mouse on Mouse Mess in IHC

    Type
    Blog Post
    ..., resulting in significant background staining (Figure 1A). Not all tissues will have the same levels ...species on species staining with no problem! But to be sure that your secondary is not binding endogenous IgGs...following three strategies might help!     Figure 1: Species on species staining can be a problem... secondary binding Direct immunohistochemistry (Figure 2A)  How do you avoid having your secondary antibody...Pre-formed primary and secondary antibody complexes (Figure 2B) Some labs have found that you can significantly...as your primary/sample, and then applying this mixture to your tissue (Tuson, et al., 1990; Goodpaster...within a problem.  Pre-block with Fab fragments (Figure 2C) Speaking of Fab fragments… Another strategy...

  5. What the HEK?

    Type
    Blog Post
    ...from? Or why they are such a commonly used cell culture model? In this blog we will spill the beans on ...postdoc Frank Graham’s 293rd experiment, hence the nomenclature we use today: HEK-293. The exact origin of the...making it genetically female. HEKs are generally cultured as an adherent monolayer, though derivatives have...However, Frank Graham showed HEKs display many immature neuron-like qualities, which is inconsistent with...cell line. What the HEK is wrong with it?   All cultured human cell lines are susceptible to contamination...immortalized, but cell health can degrade over time in culture. This can affect factors that could skew research... etc.). Thus, it’s important to maintain short culture periods (less than 20 passages) to prevent these...

  6. Hot Plasmids - June 2020 - Barcoded CRISPR Library, Sparse Cell Labeling, Calcineurin Reporter, and DNA Staining Dye Alternative

    Type
    Blog Post
    ...calcineurin activity measurements in stressed or aging cell populations. Figure 3: Micrographs of...library Article contributed by Susanna Bachle Figure 1: Initial steps in the CRISPR-pooled screening...infection (MOI) during cell library construction to ensure that the majority of cells receive one gRNA. The...Read our SPARC blog post for more details. Figure 2: Dense (c), intermediate (d), and sparse (e) ...target. Calcineurin reporters allow scientists to measure calcineurin dynamics and can serve as a drug-screening...high throughput screening, calcineurin dynamics measurements, or monitoring in vivo. To overcome these technical...dye Article contributed by Jennifer Tsang Figure 4: Double stranded DNA bound by tTALE-FP with the...

  7. Viral Genome Sequencing - A One Stop Shop for AAV Identity and Serotype Confirmation

    Type
    Blog Post
    ...10.1089/hgtb.2019.110 Pacouret S, Bouzelha M, Shelke R, Andres-Mateos E, Xiao R, Maurer A, Mevel M, Turunen...quality control (QC) mantra is to do our utmost to ensure that scientists are receiving the exact materials...substrates for tagmentation-based library preparation procedures. This finding eliminates the double-stranding...time and cost for AAV genome sequencing.  Figure 1: The viral genome sequencing workflow. Using... that looks for pre-defined serotype specific signature sequences in the data. Unlike previously described...not require any additional reagents or lab work (Pacouret, 2017). Moreover, the sequencing data from multiple...t new to Addgene, we are excited to share the procedures and Python scripts with laboratories and viral...

  8. Scientific Peer-review: Providing Critical and Kind Feedback and Advocating for Open Science

    Type
    Blog Post
    ...many iconic papers, including the ones on the structure of the DNA, were not peer-reviewed (Baldwin, 2015...the study to be replicated or omission from the literature. I think that delivering constructive critique.... The general representation of data is... The figures are ... In general, I think that this is an (e....checklist for individual sections of the paper (see figures). After finishing my section-specific comments,...WormBase, Arabidopsis.info). These repositories will ensure that the results of your colleague’s hard work ...comments will learn from them and propagate this culture further.  Many thanks to our guest blogger Magdalena...salt-stress induced responses in Root System Architecture of Arabidopsis HapMap population in Amsterdam...

  9. FPbase: A new community-editable fluorescent protein database

    Type
    Blog Post
    ...affected by environmental conditions (such as temperature, pH, fusion protein, etc...).  There are many... making such a decision remains spread across literature in publications that introduce these tools. ...references, etc...) and a more robust database structure was needed.  It is also difficult to scale such...submissions also require a DOI referencing primary literature, and every effort is made to moderate and cross-check...cross-check all data with published literature as soon as possible after submission. Using FPbase The most...’ll find matching FP suggestions. If you’re not sure exactly what you’re looking for, the Table of Fluorescent...emission spectra when available, and will soon feature filter spectra and collection efficiency calculations...

  10. Plasmids 101: How to Verify Your Plasmid Using a Restriction Digest Analysis

    Type
    Blog Post
    ...rough structure of the plasmid based on the predicted sizes and organization of different features within...and temperature compatibility when digesting with more than one enzyme. Consult the manufacturer’s manual...their defined recognition sequence. Most enzyme manufacturers make High Fidelity versions of the endonucleases...loading them. The glycerol in the buffer will make sure your sample settles in the gel well and the dyes...expected band sizes. Always run control uncut DNA to ensure your enzymes are working. When uncut plasmid DNA...approximately 75-80% of the way down the gel will ensure you keep smaller bands from running off; however...

  11. Plasmids 101: Mammalian Vectors

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    Blog Post
    ...plasmid-based research using synthetic vectors and cultured mammalian cells. Of course, these mammalian vectors...Therefore, it is not always mission critical to make sure the plasmid replicates in the cell, as many of these...foreign material and will subsequently pass it on to future generations. Stably transfected cells may be considered...stable transfection, there should be a selective pressure to force cells to incorporate the plasmid DNA ... used in conjunction with positive selection to ensure your gene gets targeted to a specific location ...293HEK, Jurkat T cells Inhibits protein synthesis; premature chain termination 1-10 ug/mL Zeocin Sh bla HeLa...is often used as a supplement in mammalian cell culture to suppress bacterial growth, and is not appropriate...

  12. Viral Vectors 101: Biosensors

    Type
    Blog Post
    ... this class measures either factors as proxies for a cellular process or directly measure a cellular process...to the structure of actinin in low vs. high tension. These systems are powerful tools to measure real-time...boiled down pretty simply – they are systems that measure biological phenomena by generating an output which...microscope, plate reader, or any other instrument which measured GFP levels and converted that signal into a readable...will focus on those which can be applied in cell culture and animal models primarily. Fun fact: Almost all...tissue preps, in multiple species, and in cell culture models. Some of them have even been expanded for...

  13. Enabling Precision Functional Genomics with the Target Accelerator Plasmid Collection

    Type
    Blog Post
    ...the amino acid change based on maps of protein structure. Sometimes this provides a great deal of clarity...bioassays could accurately test for molecular signatures of variant function and cancer phenotypes. Broad...systematically as possible. In developing these assays, we figured that they would need to be generalizable and capable...capable of measuring a variety of molecular signatures or cancer phenotypes. These assays were designed...look-up-tables” of mutant function that could enable future clinicians to improve real time decisions when ...proof-of-concept. If such approaches are proven to improve future clinical decisions, we can work together to realize...of precision functional genomics and propel the future of cancer precision medicine for the next-generation...

  14. Expanding the Targeting Scope and Editing Efficiency of Adenine Base Editors

    Type
    Blog Post
    ...passaged from an infected host-cell culture to an uninfected culture which results in less overall dilution... without DNA cleavage. Nature 551:464–471 . https://doi.org/10.1038/nature24644 Richter MF, Zhao KT, Eton...for speed: base editing before Cas lets go Figure 1: David Liu gives a talk at the Cell Symposia:...from the infected to a fresh uninfected host cell culture). This dilution step represents the major difference...dilution and less stringent phage selection. Figure 2: Phage-assisted evolution of base-editing activity...improved Cas domain compatibility and activity. Nature Biotechnology. https://doi.org/10.1038/s41587-020...

  15. Plasmids 101: Blue-white Screening

    Type
    Blog Post
    ...-peptide (represented as an orange wedge in the figure on the left) and inserted it into a plasmid, creating...note that IPTG is also included in the medium to ensure transcription of the lac operon.   Tips for blue-white...care in making your plates: X-gal is light and temperature sensitive and needs to be added to media after...autoclaving. If spread on top of pre-made plates, make sure it is evenly distributed and allow sufficient drying...analysis, like PCR or restriction digest. Make sure you use a proper E. coli strain (i.e. contains the...STBL4, JM110, and Top10 are a few examples. Make sure you use a proper plasmid (i.e. contains the α-complementation...selection methods. Combining selection and screening ensures that the white colonies you see are white due to...

  16. Dos & Don'ts When Publishing a Scientific Manuscript

    Type
    Blog Post
    ...journal. Be sure to check the Author Guidelines of the journal you are submitting to and make sure your manuscript...lab resources, such as catalogue numbers and manufacturers’ details for antibodies, plasmids (like your...F1000Research, also ask for the raw data behind the figures and results. This is a relatively new development... of science publications. Peer reviewing To make sure your manuscript is perceived as a reliable source...when publishing a scientific manuscript Dos: Make sure your paper fits the scope of the journal you are...chances of getting your manuscripts published in the future. Disregard reviewer’s comments without explaining...Always check the journals Author Guidelines to make sure that your paper fits with the journal’s house style...

  17. Antibodies 101: Fab Fragments

    Type
    Blog Post
    ...antigens.      Figure 1: IgG antibody structure with important features labeled. V = Variable ...Fab Fragment? Consider the classic IgG antibody (Figure 1): it is composed of four peptide chains (two ...different fragments that retain antigen binding (Figure 2):  F(ab')2 - These fragments are produced by...Fragment Variable (scFv). Size: ~25 kDa.   Figure 2: Comparison between a full IgG antibody and common...tool box. As you consider which tools to use, be sure to think about what pieces of the antibody you do...Bates A, Power CA (2019) David vs. Goliath: The Structure, Function, and Clinical Prospects of Antibody ...

  18. Uncertainty about Labor Law Brings More Uncertainty to Postdoc Wages

    Type
    Blog Post
    ...guest blogger, Gary McDowell, executive director of Future of Research. On December 1st 2016, many postdocs...some cases reversing these decisions entirely. At Future of Research, we had been collecting data and documenting...published in F1000Research on November 2016. Due to the nature of the postdoc position, we viewed salary raising...FLSA updates was granted nationwide. Again, we at Future of Research believe that, while the mandate has... out about what their institution is doing from Future of Research over social media, not from their institution...actually getting paid, which we will be analyzing for future updates. But suffice it to say, salaries are showing...McDowell is currently the Executive Director of Future of Research. He is interested in advocating for...

  19. Human Germline Editing Using CRISPR

    Type
    Blog Post
    ...Conclusions & implications One striking feature of this paper is the failure of the embryos to undergo editing...mutation in human embryos." Nature. Epub 02 August 2017. doi:10.1038/nature23305. PubMed PMID: 28783728. ...coverage. But the paper published August 2nd in Nature gives us even more to talk about, as it represents...but instead from the mother’s wild type allele (Figure 1). 72.4% of embryos carried two wild-type copies... proceeded through NHEJ rather than HDR. Figure 1: Homology-directed repair in targeted zygotes...broader applicability of CRISPR germline editing. Nature News mentions that the MYBPC3 gRNA used in the ...

  20. Lighting Up Cell Signaling with Photoswitchable Kinases

    Type
    Blog Post
    ...48 hours exposure to blue light also induced tail swelling in ~75% of worms (see below figure). Zhou et... current state (Green when off, dark when on). Figure 1: Photoswitchable MEK1 (psMEK1). Is it possible...is a serine/threonine kinase with a similar 3D structure to MEK1, but Raf1 and CDK5 have less similarity... pathways Negative feedback loops are a common feature of signal transduction pathways, but are often ...used a photoswitchable CDK5 in C. elegans. See figures 4B and C in Zhou et al for details on these experiments...no. 6327 (2017): 836-42. PubMed PMID: 28232577. Figures 2-4 were modified from this reference. 2. Regot...Carrasco, and Markus W. Covert. "High-Sensitivity Measurements of Multiple Kinase Activities in Live Single...

  21. Pairing CombiGEM and CRISPR for Combinatorial Genetic Screening

    Type
    Blog Post
    ...available, including those found at Addgene! Figure 1: Strategy for assembling the barcoded gRNA library...pool-clone them into the pAWp28 storage vector (Figure 1): Forward oligo: 5’- CACCGNNNNNNNNNNNNNNNNNNNGTTTGGGTCTTCGAGAAGACCTATTCXXXXXXXXC...the CombiGEM lentiviral vector backbone pAWp12 (Figure 2). Cloning from the pAWp28 storage vector to the...by NGS before beginning your experiments. Figure 2: Strategy for assembling barcoded combinatorial...and minimize off-target effects of CRISPR-Cas9." Nature biotechnology (2016). PubMed PMID: 26780180. PubMed...cells with a lentiviral CRISPR-guide RNA library." Nature biotechnology 32.3 (2014): 267-273. PubMed PMID...activation by an engineered CRISPR-Cas9 complex." Nature (2014). PubMed PMID: 25494202. PubMed Central PMCID...

  22. New Tool for Lineage Tracing: The ClonTracer Library

    Type
    Blog Post
    ...publishing, but still made it so she could “get exposure to the drug discovery process.” The ClonTracer...xenografts after cell implantation. Their goal was to figure out how to optimize protocols for in vivo screening...panned out quickly,” and was summarized in a recent Nature Medicine paper. In the publication, Bhang and coworkers...specifc, easily read sequences that can be used to measure their clonal abundance before and after a treatment... populations with resistance to both drugs. The future of ClonTracer Since starting her new role as an...populations are treated as they arise. In their Nature Medicine paper, Bhang and coworkers also looked...cancer therapy using high-complexity barcoding." Nature medicine 21.5 (2015): 440-448. Pubmed PMID: 25849130...

  23. Quick Guide to All Things Lentivirus

    Type
    Blog Post
    ...particles produced could not replicate. However, Mother Nature found a way to re-create a functional virus. Indeed...non-dividing cells is not restricted to in vitro cell culture as lentivirus-derived vectors are capable of transducing...your gene of interest into and is an important feature for your experiment; it must be chosen carefully...expression of your gene. The addition of Dox in the culture will then repress your gene of interest. This system...state and is expressed when Dox is added to the culture. This system is now used as it shows rapid gene...biosafety guidelines for more information and always be sure to consult your institution’s Biosafety Committee...cells start releasing viral particles into the culture supernatant and you can generally harvest the supernatant...

  24. 9 tips for a successful postdoctoral experience

    Type
    Blog Post
    ...workshops (see the Careers essays in the journals Nature and Science, for example). This blog post is about...write a manuscript. Consider questions like: What figures are missing? For example, are additional controls...been validated? How will the results fit into a future manuscript? You could easily spend a day or a ...experiment. However, by taking this time, you can help ensure that you understand the methodology and can have...the data and any recent relevant studies in the literature. Plus, presentations benefit from practice. If...and how your research fits in with the current literature. Finally, if you give your talk or poster to ...application and manuscript reviews, etc. You'll also meet future collaborators and get access to unpublished information...

  25. Plasmids 101: Using Transposons in the Lab

    Type
    Blog Post
    ...Transposons: The jumping genes. Nature Education 1(1):204. https://www.nature.com/scitable/topicpage/transposons-the-jumping-genes...retrotransposons are like retroviruses both in their structure and mechanism of replication. They contain two...and reverse transcriptase activities.    Figure 1: Overview of retrotransposon transposition. Retrotransposons... TEs have LTRs on both of their ends.    Figure 2: Overview of DNA transposon transposition. Modified...Transposon mutagenesis screens Transposons, by their nature, are mutagenic elements, which makes them a great...of DNA up to 10 kB in size in bacteria.   Figure 3: INTEGRATE system for RNA guided transposon insertion...Rev Genet 41:331–368. https://doi.org/10.1146/annurev.genet.40.110405.090448 Kumar A (2020) Jump around:...

  26. Plasmid-based Recombinant Monoclonal Antibodies: What They Are and Why You Should Be Excited About Them

    Type
    Blog Post
    ...  Figure 1: (Top) Polyclonal antibodies are produced in animals and consist of a mixture of antibodies...made from animals or hybridomas.   Figure 2: (Left) Figure 2: (Left) The light chain and heavy chain...Reproducibility crisis: Blame it on the antibodies. Nature 521:274–276 . https://doi.org/10.1038/521274a Begley...Raise standards for preclinical cancer research. Nature 483:531–533 . https://doi.org/10.1038/483531a Bradbury...Reproducibility: Standardize antibodies used in research. Nature 518:27–29 . https://doi.org/10.1038/518027a Bradbury... (2015) Antibodies: validate recombinants once. Nature 520:295–295 . https://doi.org/10.1038/520295b Bradbury...

  27. Plasmids 101: FLEx Vectors

    Type
    Blog Post
    ...mCherry coding sequence in the antisense orientation (Figure 1, top). The entire DNA cassette would be flanked...include both Cre and FLP recombinases.   Figure 1 (modified from Schnutgen et al. Nat Biotech 2003...identical sites on one end of the DNA fragment (Figure 1, middle). A second recombination event then excises...DNA between the identical loxP or lox511 sites (Figure 1, bottom). Since only one loxP and lox511 site...switch conditional knock-in cassette. As shown in Figure 2, the cassette should contain a reporter DNA sequence...control expression of an optogenetic reporter would ensure that the reporter remains silent until a cell or...tissue-specific Cre is provided. Since the FLEx switch ensures Cre-dependent reporter expression, the potential...

  28. Plasmids 101: Terminators and PolyA signals

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    Blog Post
    ...poly-uracil tract as shown in the figure to the right. The tertiary structure of the hairpin-DNA complex is...the transcript.   Figure 1: A predicted conserved secondary structure and sequence conserved Rho-independent...enzymes that cut RNA not protected by secondary structure. It is thought that poly(A)s are used to control...relies on a specific sequence and RNA secondary structure to induce transcript cleavage, similar to the ...motif and a downstream GU-rich region (shown in the figure below) releases the mRNA from the polymerase and...export and translation processes as well.   Figure 2: The conserved eukaryotic polyadenylation signal...

  29. Reaching out to China: Canton Nucleic Acids Forum (CNAF) 2015

    Type
    Blog Post
    ...field. Nobel Laureates and the ever expanding importance of RNA The second keynote lecture was presented...project on its 25th anniversary, how the NIH sees the future for genomics, and President Obama’s Precision Medicine...presented by Nobel Laureate Jack Szostak. Dr. Szostak’s lab continues its work on the origin of cellular life... two of the conference was kicked off by Nobel Laureate (and Addgene depositor) Craig Mello. Taking his...problem of antibiotic resistant bacteria. See the picture to the left for his list of the bacterial species...engineering. The final keynote presentation was by Nobel Laureate Thomas Steitz of Yale. He gave an update on new...conformation “not like DNA which has a boring structure.” That certainly summed up this conference—nucleic...

  30. Story of a SynBio Startup: RevBio's Epiphany (or Lack Thereof)

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    Blog Post
    ...little better. As a Master’s student, she had less pressure to publish and more freedom to attend conferences...legitimate brain exercise in developing a plan for the future. From good ideas to a SynBio startup At some point...them to see how feasible they were, conducting literature reviews and having weekly journal clubs outside...this process, we had an idea that survived the literature review, survived technical feasibility analysis... fearful of this technology and angry at the agriculture companies that produce GMO food plants. When ...idea, we knew it was a winner – although we didn’t figure out how we would accomplish the color change until...science, and to have a job that we enjoy and see a future in. And we decided to make education and outreach...

  31. Celebrating the 2024 Nobel Prize in Chemistry

    Type
    Blog Post
    ...not based on any existing protein structures, called Top7. Figure 1: Top7 receives the Nobel Prize...characterized it, producing crystal structures demonstrating “the structure is strikingly similar to the design...works and creating biological tools not found in nature. Dr. Baker’s team was the first to develop and ...wanted to create a new fold that is not present in nature (at least that we’ve seen so far!). They started...the biophysics of protein folding and selective pressures during protein evolution. The Baker Lab has deposited...

  32. Truncated gRNAs for Regulating Gene Expression

    Type
    Blog Post
    ...to Our Podcast Interview with Alex Chavez Figure 1: Schematic of native (nuclease-competent) Cas9...of genome editing (Fig. 2 and 3) (5, 3). Figure 2: Gene activation with nuclease-competent Cas9...alterations observed at the target site.   Figure 3: Gene repression with nuclease-competent Cas9... systems. She is also a NASA Fellow studying biosignature preservation in Mars analogue environments. ...proteins for RNA-guided gene regulation and editing." Nature methods 10.11 (2013): 1116-1121. PubMed PMID: 24076762... parameters via a library-on-library approach." Nature methods 12.9 (2015): 823-826. PubMed PMID: 26167643...for genome editing, activation and repression." Nature methods 12.11 (2015): 1051-1054. PubMed PMID: 26344044...

  33. No Llamas Required - Synthetic Nanobodies Against Membrane Proteins

    Type
    Blog Post
    ...antibodies that bind to the spike proteins in cell culture, and inhibit these viruses.  But llama immunization...proteins that may unfold due to camelid high body temperature (37.2 - 39.2°C). The Seeger lab found a way around...protein targets, similar to how antibodies do. The structure of the common antibody is heftier (~150 kDa) and...molecular crevices proteins on the cell surface. Figure 1: Comparison of common antibody, HCab, and nanobody...protocol, which cover 3 naturally-occurring nanobody structures (concave, loop, and convex). Each of these 3 ... to select for optimal sybody candidates. Figure 2: Outline of the process for selection of sybodies...anti-Myc antibodies bound to an ELISA plate to capture Myc-tagged sybodies. Then, the biotinylated target...

  34. AAV Vector Quality Control: Going the Extra Mile with NGS

    Type
    Blog Post
    ... like to make sure they are present at a very low concentration. So how do we make sure to catch all of...experiment because of poor reagent quality. To make sure our AAV vectors are of the highest quality, we undertake...mission of accelerating scientific research. To make sure that adeno-associated viral (AAV) vectors available...all the DNA packaged inside the viral particles (Figure 1). Briefly, packaged DNA is extracted from purified...plasmid #44362. Method validation, limitations, and future plans How did AAV-68544 become contaminated? Well...processed the mixed sample in our pipeline (see Figure 2, sample 2). When we blindly analyzed AAV-68544... the Addgene blog Check out our Viral Vectors Featured Topic Page Subscribe to Viral Vectors Blog Posts...

  35. When is a Monomer not a Monomer? The Top Three Ways Your Favorite Fluorescent Protein Oligomerizes in Cells

    Type
    Blog Post
    ...situations in cells are described below (See Figure 1).   Figure 1: Each problem with dimerizing fluorescent...follow. It's a matter of concentration Most FPs in nature are prone to dimerization (i.e. EGFP) [1, 2] or...together and can wrap the membranes into stacked structures including organized smooth endoplasmic reticulum...state, and an artificially active state due to closure by FP dimerization.   1. Transmembrane fluorescent...if a dimerizing FP is fused to the POI. These structures are large (microns), bright, non-physiological...whorl formation assay, are practical real world measures of dimerization potential that will determine ...GFP-based FRET, with application to intrinsically unstructured proteins. Protein Sci, 2007. 16(7): p. 1429-...

  36. Which Fluorescent Protein Should I Use?

    Type
    Blog Post
    ...10hours. Temperature: FPs maturation times and fluorescent intensity can be affected by the temperature. For...no use to you. When using more than one FP, make sure their emission light does not overlap in wavelength...new GFP isolated from the Unagi eel was shown to mature independently of oxygen, making suitble for use...studies (24-30°C). Brightness: Brightness is a measure of how bright is the emission. Brightness is calculated...parameters (e.g. excitation light intensity, pH or temperature). Photostability: Fluorescent molecules gets ...lose the ability to emit light) after prolonged exposure to excitation light. Photostability can be as ...parameters (e.g. excitation light intensity, pH, or temperature). pH Stability: This parameter is important if...

  37. CRISPR 101: Making a Knock-In Cell Line

    Type
    Blog Post
    ...inhibitors to your culture media prior to and after Cas9 introduction increases HDR, just make sure to remove ...distance. Homology arms and types of donors To make sure your donor molecule is used as the template, you...your Cas9 cuts in the coding region of a gene, make sure that the PAM edit you introduce is a silent mutation...exist as a repair template. To maximize HDR events, ensure cells are actively cycling through the cell cycle..., they will not undergo DNA replication, so make sure you are keeping an eye on those cells! To take this...mediates efficient homology-directed repair and termperature-controlled genome editing. Nat. Com., 8(2024...Resources on the Addgene Blog Check out our CRISPR Featured Topic Page Learn about Non-Homologous End Joining...

  38. Capturing a Moment with Marc Zimmer’s “The State of Science”

    Type
    Blog Post
    ...scientific community and their game changing nature. Full disclosure: Zimmer also spends some time on Addgene...Zimmer’s recent book, The State of Science: What the Future Holds and the Scientists Making It Happen, is an...I began my own career over 20 years ago, and I’m sure they were issues long before then. Other areas are...for me. A brief window in time “The most obvious future consequence of the undermining of the scientific...and technology.” This last section of the book, Future Science, describes the author’s thoughts on science... science and summarizing its past, present, and future in under 200 pages is an impressive task to set...Science could be a jumping off point to explore future rabbit holes. For the non-scientists in your life...

  39. Tips for Screening with Yeast Two Hybrid Systems

    Type
    Blog Post
    ...different systems could be combined effectively (3). In Figure 1 for instance, the DBD of the fusion protein shown... interest(POI’s) are fused to TAs and DBDs (see Figure 2a) (4). These fusions are then co-expressed in...DBD-POI fusion (commonly referred to as the “bait”, Figure 2b) cannot activate reporter gene expression on...promoter of the reporter gene to activate expression (Figure 2c). The reporter gene then allows detection of...the HIS3 gene, which is required for histidine manufacture and therefore growth on his-deficient media. ...known to bind serves as a good positive control to ensure that your setup works. The last thing you want ... system to detect protein-protein interactions. Nature. 1989; 340:245-246. PubMed PMID: 2547163. 5. Ozawa...

  40. Tips from the Repository Trenches: Using Barcodes to Track Samples

    Type
    Blog Post
    ... Addgene scientists inspect each sample and make sure they have been barcoded with a unique ID number ...number (1D barcode as shown below). This number ensures that every sample can be tracked easily at each step...involves good aseptic technique and controls to ensure that nothing is contaminated. We use filtered pipet...both of these are scanned into our LIMS system to ensure that they match, helping us to avoid mixing up ...glycerol stocks on Fridays. From each overnight culture, we prepare three sets of 2D barcoded tubes. Two...receiving, transforming, and storage process, we ensure that the sample tubes that we use match those of... to help track samples. They will help your lab ensure that sample ID’s are unique, consistently labeled...

  41. Making Career Development an Integral Part of the Science Training System

    Type
    Blog Post
    ...systemic biases which, in the absence of support and structure, prevent science from attracting and keeping diverse... diverse talent in science related careers. Failure to strengthen the talent pipeline sets the world up...to be successful after their training.   Figure 1: Professional Development Hub Workshop Attendees... with such a group dedicated to providing the structure and know-how to integrate career development in...areas of the scientific training and employment infrastructure. A Stakeholder Workshop at Janelia Research...2019 solidified the project and the findings were captured in this just released report “Enhancing Dissemination...attracting and launching science talent we must ensure that career skills training is a required and integral...

  42. CRISPR 101: Any Base Transversion Editors

    Type
    Blog Post
    ...included a glossary at the end of this post! Figure 1: Base transitions are edits between the purine...proof of concept for further development. Figure 2: Mechanisms of deaminase-based adenine transversion...for base editor development going forward. Figure 3: Mechanism of gGBE. Created with BioRender.com...•G base editing in mammalian cells and embryos. Nature Biotechnology, 42(4), 638–650. https://doi.org/...editor by engineered human uracil DNA glycosylase. Nature Communications, 15(1), 4897. https://doi.org/10.1038... editor with an N-methylpurine DNA glycosylase. Nature Biotechnology, 41(8), 1080–1084. https://doi.org...efficient T-to-G and C-to-G editing in mammalian cells. Nature Biotechnology, 42(10), 1538–1547. https://doi.org...

  43. Healthcare Consulting: A Door to the Business of Life Sciences

    Type
    Blog Post
    ...the success of a drug’s clinical and commercial future, and yet are incredibly difficult to find solutions...The consultants who enjoy their jobs most, get pleasure from taking up such complex challenges, quickly...parts of the job? With fast turnover of projects, pressure to maintain consistently high levels of performance...consulting can sometimes be challenging. The fast paced nature of projects often means working long hours both...using lessons from previous cases to better staff future teams. As with any highly collaborative work place... scientific background, training in reviewing literature, and critical analyses skills were applicable... and equity research firms, biotech start-ups, venture capital firms, and more. Essentially any role in...

  44. Hot Plasmids - August 2020

    Type
    Blog Post
    ...mCherry is constitutively expressed. Cells are then cultured and FACS sorted. Image from Mateo Sanchez and ...-7 Optimizing fluorescent tools for labeling structures and compartments in mammalian cells By Angela...fluorescent proteins are often used to label  specific structures, compartments, or specific localization of biomolecules...provide misleading information on where exactly a structure or protein is located within a cell. The Goedhart...encoded fluorescent markers for labeling specific structures within cells. HeLa cells were transfected...and mScarlet-I for labeling a list of specific structures within cells. For example, to improve nuclear...microtubules. Find these fluorescent mammalian cell structure markers plasmids at Addgene! Chertkova, AO, et...

  45. Supporting Reproducibility with a Connected ELN

    Type
    Blog Post
    ...tools and infrastructure in use at research universities not only today, but also in the future. Reproducibility...choosing an ELN for your research, be sure to consider how its features do and don’t support reproducibility...without access to them, the record of research captured in the ELN is incomplete. Thus, to support reproducibility...for discussion and review. It’s also possible to capture Slack conversations and import them into RSpace...and workflows that went into it. The ability to capture the conversations and associate them with a particular...ability to deposit data that has already been structured in folders or notebooks in the ELN, without having...

  46. Controlling for Off-target Effects with a New Genome-wide CRISPR Screen Design

    Type
    Blog Post
    ...Genome-scale measurement of off-target activity using Cas9 toxicity in high-throughput screens." Nature communications...this holds true in genome-wide screens.   Figure 1: Summary of the advantages of the new approach...effects of guide expression and dsDNA breaks. Figure 2: Non-targeting vs. safe-targeting guides. The...dsDNA breaks caused by CRISPR. See supplemental figure 6 for more details. Safe-targeting guides also ...of off-target cleavage by CRISPR-Cas nucleases." Nature biotechnology 33.2 (2015): 187. Pubmed PMID: 25513782...nuclease specificity using truncated guide RNAs." Nature biotechnology 32.3 (2014): 279. Pubmed PMID: 24463574... Other Resources Watch Dr. Michael Bassik’s lecture on “Multiplexing with CRISPR Screens”    ...

  47. Transferable Skills Guide: Creativity

    Type
    Blog Post
    ... everyone has their ideas heard.  So how can we assure that all participants feel comfortable sharing ...to encourage dissenting opinions is to foster a culture of inclusivity. Work with and value people who ...your lab/company/team is still working towards a culture of encouraging dissent, the next tip may be particularly...feel they can share opposing opinions as well. I am sure we have all experienced a meeting where one person...may not have the highest position or the longest tenure, but don’t let that stop you from speaking up.  ...them. If you can, set aside time to prepare for future challenges. This doesn’t have to be time-consuming...minutes every month dedicated to thinking about the future will improve your ability to innovate. (Finkelstein...

  48. 22 Hot Plasmid Technologies from 2014

    Type
    Blog Post
    ...GFPs The availability of the three dimensional structure of GFP, its brighter mutant S65T-GFP, and the ...bfloGFPc1. The plasmids used for determining the structures are now available from Addgene - bfloGFPa1 and...Mitochondrial health, transport and turnover are important features to be monitored to study mitochondrial dysfunction...dysfunction. Easy-to-use tools to monitor these features were lacking until Roberta Gottlieb and Zhen Yan labs...pTRE-Tight-MitoTimer) to show its usefulness in cell culture to report changes in mitochondrial turnover and...against common tumor markers to enhance cancer cell capture. Fierer et al., PNAS 2014 Apr 1;111(13):E1176...molecules and exceed a physiologically relevant exposure in intensity and/or duration. Both of these strategies...

  49. IBEX Knowledge-Base: A Data Resource for Multiplex Tissue Imaging

    Type
    Blog Post
    ...something doesn’t work, it’s not a failure. It’s an education.   Figure 4: Zenodo page showing the IBEX...reagents that work for our experiments!)     Figure 1: The IBEX Imaging Community front page. That...both their successes (recommended reagents) and failures (not recommended reagents) along with information... directly from the Zenodo dataset.     Figure 2: Screenshot showing reagent entries with associated...discussion with others in the IBEX community.   Figure 3: Screenshot showing recent topics in the IBEX...method for high-content imaging of diverse tissues. Nature Protocols, 17(2), Article 2. https://doi.org/10.1038...

  50. Viral Vectors 101: Integrase-Deficient Lentiviral Vectors

    Type
    Blog Post
    ...here and no viral progeny are produced (Figure 1).  Figure 1: Comparison of the life cycle of a lentiviral...recombination or non-homologous end joining (see Figure 2).  Figure 2: Schematic representation of integration...cell but cannot integrate it into the host genome (Figure 1). IDLVs were first discovered by researchers ...continuously expressed (Fu et al., 2013). Using IDLVs ensures transient expression without the risks associated...titering IDLVs, there is no integrated piece to measure! Instead, two commonly employed methods for titering...induced by CRISPR-Cas nucleases in human cells. Nature Biotechnology, 31(9), 822–826. https://doi.org/...
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