... of these methods to ensure your insert retains the 3' A overhang: Use a mixture of proofreading enzyme...restriction enzymes or ligase, and requires no post-PCR procedures. Sounds easy right? The technique relies on the...how does TOPO cloning work? As illustrated in the Figure below, the “A” overhang on the blue PCR product...insert, the magic of TOPO cloning happens. Basic procedure Let’s break down the steps needed for TOPO cloning... TOPO Vector.  3. Incubate 5 Minutes at Room Temperature: You can place your reaction on ice if you are...extension time after the last cycle of PCR to make sure that the "A" gets added to all PCR products. Keep...electroporation to fail). When incubating at room temperature, it is not recommended that you exceed the 5 ...

...assays, such as ELISAs and Westerns, frequently denature the proteins as part of the sample prep. This ...it from its native state to a less folded, or denatured, one. This enables absorbing of the proteins to... or ELISAs may therefore often be specific to denatured versions, or even to just that particular assay...    Fig. 1: A native protein (left) is denatured, allowing the antibody to bind to a previously...you’re working with native proteins instead of denatured ones - well, don’t relax just yet. Proteins often... state or specific configuration. So how do you ensure that you have the right antibody for the protein...Did you have a denaturing step? Do you want to capture the protein bound to anything? Is it a transmembrane...

...marker and origin of replication. During bacterial culture, only the plasmid with the antibiotic resistance... that contain the full length plasmid.   Figure 1: The full lentiviral plasmid is expected to be...researchers find that using a 30°C incubation temperature to grow bacteria more slowly helps reduce the...Esposito (2018) noticed that in Stbl3 bacterial cultures with lentiviral vectors, the flat white colonies...tan colonies that look similar to normal E. coli cultures contain the recombined vector backbone. Test multiple...multiple colonies before saving a glycerol stock to ensure the colony you selected contains the full plasmid...length plasmid. If you find that your bacterial cultures contain a mix of full length plasmid and the recombined...

...mushroom escapes US regulation. Nature 532:293–293 . https://doi.org/10.1038/nature.2016.19754 Zhang H, Demirer...Fan C, Landry MP (2019) DNA nanostructures coordinate gene silencing in mature plants. Proc Natl Acad Sci...sufficient to modify the genome of plant cells. The structure of the gRNA (composed of a ~20 nucleotide targeting...components is essential for any CRISPR experiment, and failure to express either the gRNA or Cas9 in your cell...into a target cell line or organism (Presented in figure 1). More information on Agrobacterium-mediated ...mediated cloning of your gRNA of interest. Figure 1: Simplified schematic of agrobacterium-mediated... (carbon nanotubes, Kwak et al., 2019), DNA nanostructures (Zhang et al., 2019) or cell-penetrating peptides...

... not being interviewed, there could always be a future boss in the room, so it is a good idea to start...academic position, see this very good article at NatureJobs: Interviews: The All-Important Job Talk. 1. Watch...Scientists do this all the time, you just need to figure out how to make it suspenseful. 4. Start preparing... Google.slides or Powerpoint file right now to capture interesting key results, striking images, creative...Of course, if some of your work does relate, make sure to include that material, but don’t force it. You...explaining how your work fits in the bigger science picture. Scientists especially like to learn new things...resulted from their research, “aha” moments, and future research directions. Don’t worry if your work hasn...

...our journal-adjusted citation measure we re-weight the 3 percent of Nature papers without Addgene plasmids... (=C*G) 1 2010 3 Nature  3.3% 10.5%   3.2  9.6 2 2010 167  Nature 3.3%  10.5%  3.2  534.1...also pulled the big interdisciplinary journals: Nature, Science, PLOS One, and PNAS. We then broke the...papers without plasmids deposited with Addgene (Figure 1). Even aggregated this much, you can see the... Addgene plasmids in our data were published in Nature, but only 3 percent of papers without Addgene plasmids...papers with and without Addgene plasmids are in Nature. This is also true for other journals, so our estimates...Environmental Microbiology  1.6% 1.2%  0.75   125.3 In figure 2, you can see the full impact from using these...

...Austen’s Pride and Prejudice, and some more obscure treasures, like Tove Jansson’s The True Deceiver. There...on-site book swap/library, reading is part of the culture here at Addgene. Since it’s National Book Month...own hands. Who doesn’t love a book series that features a young female protagonist, some clever magic,... may be intimidating, Ruthefurd’s writing will capture the attention of even the most skeptical reader...club Indeed, reading is ingrained into Addgene’s culture. We have a book club in the office that was founded... had a few reasons for launching this one. On a purely selfish level, I wanted to be challenged to read...has become one of my favorite parts of Addgene’s culture. Have you read any of these books? Whether you ...

...absolutely possible to use CRISPR to cure genetic disease in the near future,” Li says, though there are some... derived from iPSCs. From Rohn et al. 2018; see figure 3 for full details.    CRISPR advances in monoallelic...receptor (CFTR) by homologous recombination in cultured intestinal stem cells of patients with cystic ...When all was said and done, the researchers had cured 24 mice of their disease. Meanwhile, Hans Clevers...because the genomes of adult stem cells in organoid cultures are stable,” said Gerald Schwank, first author...off-target effects is a major limitation. The procedure will also need to be perfected until the efficiency...of repair approaches 100 percent. “We need to be sure it's accurate enough and efficient enough for repair...

...spectrophotometer measures these absorbances using UV-transparent cuvettes. First you measure the absorbance...in. This is a “blank” and measures the background absorbance. Then you measure the absorbance of the DNA...DNA sample.  These absorbance measures give you an idea of the concentration of your DNA prep and whether...Generally, A260 of 1.0 is equivalent to 50 ug/ml pure dsDNA. Use the following formula to estimate your...as compared to spectrophotometric methods that measure all nucleic acids). These methods are more sensitive...quantified first before larger fragments. Figure 1: The quantification of a RNA ladder using capillary...sensitivity so it’s not used much. However, the measurements are made in the visible range and can be read...

...strand in Figure 1 below) was free to anneal to complementary ssDNA molecules in vitro. Figure 1: Cas9...presumptive mechanism of action are presented in Figure 2. Figure 2: Single-stranded donor DNA interaction...Understanding Cas9 biophysics enhances HDR We first measured Cas9 dissociation rates and discovered that the... of approximately 6 hours. To our surprise, we measured identical dissociation rates for catalytically...catalytically inactive dCas9 produced a “bubble” structure in uncut target DNA that was accessible to anneal...single stranded DNA. We thus wondered if such a structure could drive sequence replacement in cells. Targeting...inactive CRISPR-Cas9 using asymmetric donor DNA." Nature biotechnology (2016). PubMed PMID: 26789497.  2...

...make sure the work gets done and done well. Involving your team effectively is now your new measure of ...Setting someone up for failure by delegating is a bad idea. How do I make sure it goes well? As with ...appropriate communication between members of the team to ensure that no component of the overall project gets left...fun, but getting buy-in when delegating can help ensure successful results.  Can you provide the information...will be made that could have been prevented.  Make sure everyone understands that no question is “dumb” ...and plan changes. Written documentation helps make sure everyone is on the same page. Effective delegation... the best use of your time and skills. It also ensures that other people in the team grow and develop ...

...brightness in a system closely mimicking the future application. In figure 2 we provide an example of the comparison...spent and, if you do it right, can be (part of) figure 1 of your next paper or thesis. This series of ...protein is based on the properties found in the literature or tabulated data that summarizes properties ... and therefore high brightness is an important feature of a fluorophore. The key question is how the brightness...detector sensitivity) and the biological system (temperature, prokaryote versus eukaryote, background fluorescence...within the dish. This is caused by the stochastic nature of plasmid uptake by cells. To correct for this...2010). An explanation of the assay is depicted in figure 1. The strict correlation between the fluorescent...

...interactions. ChIP works by using antibodies to capture protein:DNA complexes with antibodies specific ...your protein(s) of interest. Once the complex is captured, it is treated so that the protein and DNA are...Once you have your sample, the first step is to ensure your protein:DNA complexes will remain bound to...size, typically between 200-1000 bp. This is to ensure that your downstream analysis focuses on the DNA...the sample, introduces bias due to the selective nature of the cutting. While sonication is usually the...DNA and then determine concentration.  Protein Capture After crosslinking and sonication or enzymatic ...native proteins (so antibodies validated only for denatured proteins may not be a good choice) and the amount...

...process led to quick dives back into the literature to make sure absolutely everything was correct. Final... we usually make figures in BioRender or use appropriately licensed and cited figures from papers or image...content - the useful information on scientific literature, technical skills, and experiences from thousands... Nearly everything we write entails a primary literature search to collect up to date information on the...Final touches We try to use figures and images to help illustrate our points and break up walls of text...graphic designer made most of the Antibody Guide figures. This requires a fair amount of communication between...our content team and our graphic designer to make sure things are clear, accurate, and in line with Addgene...

... unable to secure enough money to sustain a startup, he went to work for Flagship Ventures – a local Cambridge...in academia and industry vary drastically, he reassures that one can learn it by reading a broad variety...of materials and constantly practicing. “The expenditure of effort in preparation and practice is directly... Cambridge biotech venture capital firm with deep ties into the local academic community. However, Mike...entrepreneur must be OK with the constant fear of failure at all stages in the startup". To gain insight ...and vision for the company; this will help avoid future complications and potential conflicts that waste...takes around 1 – 2 years. The product is the measure of success. Technology is proven in (A) Productize...

...about are (1) your experimental conditions and measurements; (2) the cells you’re targeting; (3) how much...vector: serotype, promoter, and titer.     Figure 1: Flow chart depicting a) steps for selecting ...injection site than AAV2 (Watakabe et al., 2015) (Figure 1). You’ll need to consider if such properties ...goals when designing and testing your AAV.  Figure 2: Comparison of AAV serotypes 1, 2, 5, 6, 8, and...regions.  Image modified from Aschauer et al., 2013, Figure 1. Used under Creative Commons Attribution License... 2004; Taschenberger et al., 2017).     Figure 3: Comparison of two astrocyte selective promoters...neurons (NeuN). Image from Griffin et al., 2019, Figure 4. Used under Creative Commons Attribution 4.0 ...

...between strains of one species, so be sure to check the literature and validate your choice of AAV vector...adeno-associated virus infection. Nature, 530(7588), 108–112. https://doi.org/10.1038/nature16465 Rubin, J. D., Nguyen...allowing them to target particular cell types. Figure 1: Diagram of AAV components. Created with BioRender.com...vectors have both distinctive capsids and genomic structures. The viral capsid is the key that lets AAVs “...mixing, matching, and modifying capsid and genomic structures. For example, rAAV pseudotypes mix the genome... by Addgene are pseudotypes using the genomic structure of AAV2, so they are formally called rAAV2/1, ...and so on, although we often use abbreviated nomenclature (AAV1, AAV2, etc.) to distinguish the capsids...

...to the system and in what order they were added. Figure 1D below demonstrates an example of a register ... in certain states of interest. For example, in Figure 2 below we present a two-input, five-state GRSM...we implement RSMs in E. coli with two plasmids (Figure 3 below): Input Plasmid: The input plasmid (high... RSM (See performance data from the indicated figures in our paper (1)) Input Plasmid Output Plasmid ...designed to have 20-30 nt length, ~60 oC annealing temperature, and ~50% GC content) so that individual parts...Ara) RSM over a time series of increasing input exposure lengths and found that two hour inductions were...each possible permuted substring of inputs (see Figure S9 of our paper (1)). We anticipate that, depending...

...are doing multiple digestion be sure that the buffers and temperature are compatible between the different...or use a different temperature (30°C) for the growth on plates and in liquid culture. If you don’t get ...mindful of the restriction enzymes you chose. Make sure that enzymes that you are using are not blocked ... few general parameters that you can adjust: temperature and time of incubation, and amount of DNA. As...a few colonies (5-10) and grow them in a small culture volume (2 or 3 ml) containing the corresponding...following steps: Do a single or double digest to be sure that the plasmid is the correct expected size and...acknowledged in hundreds of publications including Cell, Nature, and other journals.   Paolo Colombi is currently...

... achieved and the opportunities we have for the future. At Addgene we will never ever stop planning, perfecting...re meeting the challenges of DNA distribution to ensure scientists everywhere can access plasmids despite... team. We show up in both science journals like Nature and in mainstream media like The Atlantic. We’ve...working in lock step to maximize the services and features we can offer online for Addgenies and to serve...Addgenies strong. The Addgene Board of Directors is mature and poised to support our growth. Thanks to Joanna...thinking, and openness to team work.   Company culture Once in awhile I worry a very little bit about...Best Place to Work with a positive, supportive culture, we are attractive to the local employee pool and...