...Transgenesis." PLoS ONE. 8(12): e83043 (20 December 2013). 2. Carola Engler et al. "A One Pot, One Step, Precision...Lohmann said they’ve only tested GreenGate in Arabidopsis, it should in principle work in any plant species...

...First, the light-responsive molecules of cryptochrome 2 are the chromophores flavin-adenine dinucleotide and...ingredients - CRY2 and CIB1 – which they borrowed from Arabidopsis thaliana. The results are light-inducible transcriptional...

...six different research constellations: Doench/Root (2), Zhang (3), Wu (4), Yusa (5), Wang/Lander/Sabatini...Bioinformatics 33.7 (2016): 1099-1100. PubMed PMID: 28414855. 2. Doench, John G., et al. "Optimized sgRNA design to...single cell level), are separated by, for example, cell sorting. The separated cells are subsequently ...which gRNAs are differentially expressed in the separate groups and, as a consequence, which genes are ...the protein following stimulation would then be separated by sorting, and subsequently sequenced to identify...suggested gRNA sequence including adapters. Output_UserInputParams: Contains all the information that you ...al. "Unraveling CRISPR-Cas9 genome engineering parameters via a library-on-library approach." Nature methods...

...teaching and mentoring others (see more in Figure 2). Starting early demonstrates not only your interest...application process so you can learn more! Figure 2: The many ways to prepare for a science policy career...the work you do to creatively connect seemingly disparate individuals and ideas toward solving one problem...evidence-based university policy to combat sexual harassment in academia or a large company who decides how...

...another. You’ll need to validate that points 1 and 2 hold true under your specific experimental conditions...when (1) other strategies may not be feasible and (2) large-scale proteomic and transcriptomic datasets...antigen - Recognition of an immunogen does not guarantee recognition of the target antigen. You want to... transfect it into your favorite cell line. In parallel, you transfect cells with another plasmid not ...

...make it easier to study these mutants.   Fig. 2: (A) Betacyanin is produced from tyrosine by the activity...occurs in T0 plants (from callus) or T1 plants (Arabidopsis floral dip). In the next generation the CRISPR...planning to use it to simplify my work with an Arabidopsis mutant that can’t set seeds. I study the development...   Fig. 3: (Top right) A RUBY-expressing Arabidopsis seed following floral dip transformation next...without antibiotics. (Top left) Mature silique (Arabidopsis fruit) with fertilized developing seeds from ... the Plant Genome Using CRISPR/Cas9 Tips for Arabidopsis Transformation CRISPR 101: CRISPR-Mediated Plant...

... stabilization of unknown bacteria. Figure 2: Christina Mork collecting samples at the hot spring...microfluidic parallelization to sequence this highly complex hot spring sample. The sample was separated into ...Quake Lab member joined for half of the trip. Preparations ahead of time included contacting YNP staff ... Paul and Anastasia (Potanina) Neddersen characterized a few samples by 16S rRNA gene sequencing which...thermostable Cas9 enzymes have previously been characterized: GeoCas9 from the Doudna lab and ThermoCas9 ...

... ligands can elicit varying degrees of β-arrestin-2 recruitment to receptors, indicating their agonistic...fluorescent labels. In Nature Methods (Vol. 19, Issue 2, pp. 149–158). Nature Research. https://doi.org/10.1038...help. Here, we will outline some fundamental characteristics to consider when selecting a dye or ligand ...Fluorescence microscopy takes advantage of the characteristics of these fluorophores to enable highly specific...receptor conformation and signaling activity​​ (Sridharan et al., 2014). It's also crucial to evaluate how...impacted by both the excitation coefficient, characterizing the strength a species absorbs or reflects ...considerations regarding the choice of fluorescent dyes (Sridharan et al., 2014). Manufacturers will typically recommend...

...phosphoinositides (Varnai and Balla, 2008) (Figure 2). RNA production can be detected by using a MS2 coat... 296.5569 (2002): 913-916. PubMed PMID: 11988576. 2. Tsien, Roger Y. "The green fluorescent protein." ...microscopy (University of Amsterdam). He develops, characterizes, and uses genetically encoded fluorescent probes...

...containing complementary polynucleotide chains. J Virol 9(2): 394-396. Lecomte E, Tournaire B, Cogné B, Dupont...facilities producing a large number of viral preparations in parallel, there is a very real risk of mixing up...Léger A (2015) Advanced Characterization of DNA Molecules in rAAV Vector Preparations by Single-stranded Virus...confirm the identity and serotype of our AAV preparations. We recently published a description of the ... For labs that produce a small number of AAV preparations this level of quality control is sufficient ...AAVs and the TA-based ligation and tagmentation preparation approaches described previously require a preliminary...effective substrates for tagmentation-based library preparation procedures. This finding eliminates the double-stranding...

... of my favorite molecular biology tool, P1 phage. 2) The post describes an ingenious but simple technique...That’s it for now. As I indicated in the opening paragraph, I’m currently on my way back from a very successful...

...method relies on the fluorescence ratio between the 2 fluorophores, rather than on the fluorescence intensity...are also known to trigger seed germination of parasitic plants, this approach was also used to build and...ratiometric fluorescent biosensor based on the parasitic Striga hermonthica SL receptor HTL7. This novel...compounds that have the potential to regulate the parasitization of cereal crops. As a result, agricultural ...

...in just 2 years and excitement about how much more there is left to do with the characterization and implementation...relatively isolated resorts creates a sense of camaraderie with fellow attendees from the moment you arrive...motto for scientists who contact us and want a guarantee that their experiments will work (yes, this happens...

...interferon gamma (IFNγ) - and eliminate cancer cells. 2. Equilibrium - In a process analogous to the development... 35. Academic Press, 2015. PubMed PMID: 25818339. 2. Curiel, Tyler J., et al. "Specific recruitment of...thanks to our guest blogger Subhadra Jayaraman! Subhadra Jayaraman is currently a doctoral candidate at...post was contributed by guest blogger Subhadra Jayaraman, a doctoral candidate at Binghamton University... level of progression, anatomical location, characteristic (solid localized tumors or systemic tumors)...

...establishing the dynamic range of your FRET assay (Figure 2). These labeling methods are freely interchangeable...PMID: 20161833. PubMed Central PMCID: PMC2802762. 2. Dumas, Anaëlle, et al. "Designing logical codon reassignment–Expanding...), quantifying antibody/antigen interactions (Saraheimo et al., 2013), and visualizing structural changes...fluorophores, it’s best to consider these cases separately when talking about the tools that are available...tools for labeling reagents and structurally characterizing large ion channels involved in skeletal muscle...40.3 (2006): 279-286. PubMed PMID: 16908183. 10. Saraheimo, Satu, et al. "Time-Resolved FRET-Based Approach...

...from a single Illumina MiSeq® run. As shown in Fig. 2, below, differences in input concentration across ...many labs. However, the high costs of sample preparation for high-throughput next generation (NGS) sequencing...Addgene to develop and apply our plexWell™ Library Preparation Technology for NGS-based sequencing and confirmation...of barcoded fragments is known as a *library*. Parallel sequencing of the fragments within a library allows...understated problem in multiplex NGS library preparation. The challenge here is that, as more samples ...minimize these issues, researchers usually include a separate normalization step where individual input DNA ...

...PMID: 25386923. PubMed Central PMCID: PMC4227841. 2. Young, Carissa L., Zachary T. Britton, and Anne S...number of disulfide bonds in your protein - two parameters that affect how well E. coli can produce your...

...development and therapy. Nat Rev Drug Discov (2017) 16(2):89-100. DOI: 10.1038/nrd.2016.238 Additional resources...drugs. While we often think of these two fields as separate, they are deeply intertwined. In this blog we ...Target identification Not all diseases are fully characterized or have a simple pathology. In these cases, ...

...access this tool, set up an IDT account. Figure 2: Addgene's Analyze Sequence tool.   DNA sequence...complement sequence.  Filter DNA tool: Removes non-DNA characters, such as numbers and blank spaces, from the text... primer dimer estimation, and other primer characteristics. NEB Data-optimized Assembly Design (DAD): ...

...motion videos is very time consuming; producing a 1-2 minute video takes an entire week of our free time...gotten easier now that I identify with the main character in the video. We’ve grown attached to the work...“WOW, now I have my own laboratory” (the main character is much more than a piece of plastic for us now...

...incorporation of biosensors, protein evolution lineages, 2-photon spectra, more elaborate spectrum analysis tools...complicated entities with a large range of characteristics (color, brightness, photostability, maturation...attach important references that serve to further characterize a protein of interest (and we very much appreciate...simplifies the process of building advanced multi-parametric search queries.  Looking for a bright, monomeric...emission peak around 600nm?  Simply input these parameters and you’ll find matching FP suggestions. If ...

...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... a mouse model, and its success is not always guaranteed. To learn more about this process check out our...

...), 1668. https://doi.org/10.1038/s41467-019-09656-2 More resources on the Addgene blog CRISPR101: Validating...plasmid that leads to higher titer lentivirus preparations than the first generation CHIME (pXPR_053). ...

...photoconvertible fluorescent protein. Nat Methods. 2009 Feb 6(2):131-3.  PubMed. Find plasmids from this publication...optogenetics. Even though GFP has been so thoroughly characterized, it turns out this protein has a few more secrets...

...E. coli: acoustic gene 1 (arg1) and acoustic gene 2 (arg2). The key difference between arg1 and arg2 vesicles...genes for in vivo imaging Following in vitro characterization, Bourdeau et al performed a proof-of-concept...

...Genome Editing and The CRISPR Software Matchmaker Day 2: Homology directed repair: CRISPR 101: Homology Directed...but with far fewer off-target effects! This characteristic probably explains why pC016 - LwCas13a guide...

... enrich the microvesicles containing AAVs (Figure 2). Since the development of the exo-AAV isolation protocol...production phase. However, this has not been well characterized as it is not a common event. What are exosome...exo-AAVs rely on differential centrifugation steps to separate cell culture supernatant containing exo-AVVs from... when compared to the conventional AAVs (AAV preparations were always normalized to their genomic titers...29296848. PubMed Central PMCID: PMC5728288. Schiller, Lara Timantra, et al. "Enhanced Production of Exosome-Associated...2018): 293-305. PubMed PMID: 29767307.  Wassmer, Sarah J., et al. "Exosome-associated AAV2 vector mediates...

...improve the brightness of previous Nano-lanterns by 2-4 fold and permit five-color fluorescent imaging! ...expressed in cells, generates much less signal than comparable fluorescent proteins. This weak emission hasn...! These eNL constructs use the brightest characterized luciferase, NanoLuc, and its substrate furimazine...Nagai laboratory has a done a wonderful job characterizing Nano-lanterns and eNL for many different applications...Nano-lanterns requires a linear unmixing algorithm to separate the various colors. In fluorescent imaging, you.... Filtering the emission wavelength partially separates the signals of various Nano-lanterns, but it does...

...types within a sample to expected labeling (Figure 2). The panel of antibodies used in this approach should...the antibody in the same tube.     Figure 2: Schematic showing the use of an orthogonal approach....   Once an antibody is found that shows separation between the wild-type and the KO population (...optimization can be performed to both maximize the separation and reduce any background staining. This can ...for a flow antibody, the YCharOS (Antibody Characterization through Open Science) initiative has now started... started producing flow cytometry characterization data for selected antibodies, using the knockout approach...antibody can be assessed through its ability to separate the cell lines based on expression of the target...

... tabular HTML formatted output. Surprisingly only 2 tools provide FASTA output. A few notable tools allow...Nature biotechnology(2014). PubMed PMID: 24880488. 2. Hsu, Patrick D., et al. "DNA targeting specificity...years ago I was a part of a group (Biology of Host-parasite Interactions, Institut Pasteur, Paris) that changed...editing the genome of Plasmodium falciparum (the parasite that causes malaria). I attribute their success... is, however, not a focus of this blog post. I separate sgRNA design tools into database and de novo solutions... With respect to de novo sgRNA design, I again separate it into two categories. On the one hand we have... al., 2013, other tools have incorporated new parameters such as the Doench-Root score (by Doench et al...

...peer-mentoring groups at Addgene have gone virtual!   2. Practice public speaking For almost any science career...points for being able to just look at a paragraph and do a character count in your head.  Here are some sites...

...PMID: 30083608. PubMed Central PMCID: PMC6070312. 2. Stark, Jessica C., et al. "BioBits™ Bright: A fluorescent...school classrooms, like gel electrophoresis to separate DNA or transforming cells with green fluorescent...in functional genomics research Learn how the Arabidopsis research center brings plant science to life ...

... 293HEK Inhibits termination step of translation 2-10 ug/mL G418/Geneticin neo HeLa, NIH3T3, CHO, 293HEK...called transfection. Transfection is somewhat comparable to bacterial transformation (the introduction... a problem depending on the duration or other parameters of your experiment. Mammalian cells double at...reduces the rate of plasmid dilution but does not guarantee 100% transfection efficiency. Stable transfection...

...create a recombinant antibody.     Figure 2: Changes to the heavy chain or light chain genes within...the antigen-binding sites of the antibody - the paratopes - may change and impact the specificity and avidity..., Aldred SF, Bestagno M, Burrone O, Maynard J, Ferrara F, Trimmer JS, Görnemann J, Glanville J, Wolf P...Versus Polyclonal Antibodies: Distinguishing Characteristics, Applications, and Information Resources. ILAR...

..., N. and Hamilton, D. 1981. PubMed PMID: 6276558. 2. Site-specific DNA recombination in mammalian cells... it, allowing for two separate recombination events to be controlled in parallel. Have you used the Cre-lox...consisting of two 13-bp long palindromic repeats separated by an 8-bp long asymmetric core spacer sequence... which can be located either on the same or on separate pieces of DNA. Both 13bp repeat sequences on a...forming a dimer. The two loxP sites then align in a parallel orientation, allowing the four Cre proteins to...maintained).  Translocation: If the sites are on separate DNA molecules, a translocation event is generated...Cre into cells of interest. What if I need two separate recombination events? One potential limitation...

...light sensing proteins. Furthermore, during the first 2 weeks of life, larvae already exhibit distinct behaviors... the escape response neural pathway is well characterized, there is much left to be discovered of spontaneous...

...project my team & I have poured our hearts into for 2 yrs) has 200K+ figures by 95K+ scientists 👨🏽‍🔬👩‍...New icons can be requested, although there’s no guarantee Biorender will create it. Additionally, you can...

...the world’s population, we account for an estimated 2% of global plastic generation. Less than 9% of plastics... References Geyer, Roland, Jenna R. Jambeck, and Kara Lavender Law. "Production, use, and fate of all ...

...affinity and specificity. Trends in Immunology, 29(2), 91–97. https://doi.org/10.1016/j.it.2007.11.004 ...kill the target cell. If the pathogen is a large parasite, ADCC can be induced through a very similar interaction...

... master's degree prepared them for their career.  2. Are you looking for career advancement and growth... conferences, help with manuscript and grant preparation, and lead your own research project.  3. Do you...

...easily understood and consistent in design and layout. 2. Be concise in your writing  When writing a manuscript...worthless. But simply writing down your data doesn’t guarantee a publication. There is an art to composing manuscripts...

...but also something about how science really works. 2)   Read science writing in magazines or newspapers...people like a good story and science involves characters – in this case you – on a quest to ask and answer...

...articles, such as: Sander JD & Joung JK, Nature Biotech, 2 March 2014. Or browse the articles related to the ...system, developed by the Zhang lab, which has comparable efficiency to the optimized chimeric design but...

...PMID: 30356044. PubMed Central PMCID: PMC6200774.   2. Nielsen, Alec AK, and Christopher A. Voigt. "Deep...properties (GC content or rare codon percentage) and comparative properties (percent sequence identity and percent...

...backbone, which expresses the gRNA from a Drosophila U6:2 promoter and Cas9 from the actin 5C promoter. Addgene...backbones for lentivirus LV 1-5 and retrovirus RV 2-5 were constructed. The authors then used GMAP assembly...engineering (MAGE) (1). MAGE uses recombineering (2) to simultaneously incorporate multiple single-strand...number of tandemly expressed gRNAs respectively) and 2) the size of the deletion. Smaller deletions generally... Kroeze et al. developed a new approach termed Parallel Receptor-ome Expression and Screening via Transcriptional...library of approved drugs against the entire kit; a parallel analysis that successfully identified new, highly... generated using slightly relaxed gRNA design parameters. To demonstrate the effectiveness of their library...

...acceptable variations in DR sequences. Figure 2: comparison of Cas 9 vs. Cas 12a expression cassettes...Cas12a’s ability to knock out multiple genes in parallel. Lastly, this Cas12a-based screening method was...gels. A: individually expressed proteins run in separate lanes. B: individually expressed and coexpressed...containing 150 or 250 kD proteins are also available separately. The proteins, which are purified via histidine...or Cas12a. Despite its small size, CasMINI is comparable in efficacy to dCas12a systems in both gene activation...

...closely mimicking the future application. In figure 2 we provide an example of the comparison of the practical...your selection on a small-scale head-to-head comparative study under conditions that best mimic the intended...practical brightness takes all application specific parameters into account, including the specs of your microscope...translated from the same open-reading frame and separated from the protein of interest by a 2A self-cleaving...microscopy (University of Amsterdam). He develops, characterizes and uses genetically encoded fluorescent probes...

...higher quenching of mScarlet emission. Figure 2: Ace-mScarlet is a fusion between the voltage-sensitive... was also between 1013 - 1014 vg/kg, a dosage comparable to those currently used in human gene therapy...response and brightness. They also do not spectrally separate from green sensors and blue-light-activated optogenetic...phytochrome-like fusion proteins from the plant Arabidopsis thaliana. In the presence of red light (650 nm... PCB is no longer present, the fusion proteins separate, inactivating Cre. This is critical for reducing...

...responsible for that differentiation.   Fig. 2: Single cell RNA-seq clustering of cells transduced...road map, and what was known about previously characterized TFs, the Zhang group were able to classify unknown...studied TFs, this functional association of the uncharacterized TFs is a useful finding. Transcriptional program...

...cellular physiology.  https://doi.org/10.1002/jcp.30064 2. Barman et al. 2020. “A glance at genome editing with...with a dominant mutation in a gene that causes cataracts could be rescued by coinjection into zygotes of... explore its potential in mice with a dominant cataract disorder caused by a single copy of a gene known...