... pCAG-T7-TALEN(Sangamo)-Destination series, pCAG-Golden-Gate-Esp3I-Destination Takashi Yamamoto pcDNA-TAL-NC2...they used CRISPR to create OsTIR1-expressing cell lines by inserting OsTIR1 into the "safe harbor" AAVS1...Recent studies carried out by Sebastian Lourido and colleagues constitute an important step toward understanding...Alexander Stark and colleagues at the Research Institute of Molecular Pathology have developed an alternative...publication, Kroeze et al. describe their enhanced TANGO arrestin recruitment assay incorporating 314 codon-optimized...bacteria, the process of cloning, combining, and testing these different parts can be quite arduous. The...your gene of interest and cloned into provided destination vectors using low-cost, one-pot golden gate cloning...

...ordering food - everything is covered.   So why are we still printing and gluing pieces of paper in notebooks... needs The familiar paper-based lab notebook is still a standard approach to record-keeping in many labs...In their ideal form, ELNs provide users with a holistic means to manage their work, combining data, team...researchers. The full story is available here. Our questions focused on researchers’ current practices, their...maintenance and support.” Besides that, academic institutions tend to think about the long term, so they need... once they establish the system within their institution. In the study, respondents were also asked about... knowledge of electronic lab notebooks into 9 questions that will help you start and define what exactly...

... order.  Still have questions? Visit the Addgene Help Center If you have additional questions about our... my order? All of these questions are really getting at one bigger question: How does a customer place...answers to other ordering questions here. In addition to answers to common questions about the ordering process... discussing some of our most frequently asked questions related to the ordering process: I placed an... purchase order, which is an official document listing materials and pricing information that is sent ...repository. We distribute it on behalf of depositing institutions. Addgene cannot fulfill orders based on PO documents...

...at allencell.org and the Allen Institute website at alleninstitute.org. A classic challenge in cell biology...at allencell.org and the Allen Institute website at alleninstitute.org. References 1. Roberts, Brock, ...contributed by the gene editing team at the Allen Institute for Cell Science. Learn more by visiting the Allen...large tags into a precise location in the genome is still inefficient, particularly in human cell lines. Furthermore...genomic tagging experiments. Browse the Allen Institute for Cell Science Plasmid Collection Fluorescent...edited hiPS cell lines generated using the Allen Institute for Cell Science Plasmid Collection, please visit...Many thanks to our guest bloggers from the Allen Institute for Cell Science. This post was contributed by...

... a function of digestion time. For phage DNA that is refractory to enzymatic digestion, however, a physical...diverse biological entities on our planet, but we still know collectively little about how they infect and...RBPs, the host recognition factors of phages) constitute one of the most popular classes of phage proteins... the process of capitalizing on phage RBPs as diagnostic tools and therapeutics. In addition, the strategic...recognize on a host cell. To make matters even more interesting, much of what a phage faces as it approaches ...library of phage DNA This first step involves digesting phage genomic DNA into 1-3 kb (approximately gene-sized...units, which can be purchased cheaply, are small plastic containers that can easily be attached with a hose...

... do during your training is meet interesting people doing interesting things. I call this building relationships...will be asking the questions, so be prepared. There are many good lists of questions on the internet. To...learning new things from other scientists doing interesting work. Consider this to be like any other research...mind that this is a good time to ask personal questions about salary, opportunities for advancement, challenges...challenges and disappointments. These more personal questions can elicit the most valuable insights you will...people like to help One good place to look for interesting connections is your university’s alumni database...too busy to respond to you). You will uncover interesting opportunities by meeting with people working ...

...last week.  This fantastic city-wide effort was led by two amazing postdocs, Kristin Krukenberg and Jessica...oft quoted statistic that only 15% of current postdocs will be academic principal investigators. She also...for science—we have powerful tools, beautiful questions, and smart scientists.  At the same time, he pointed...He charged the postdocs to “Grab ahold of your destinies” and also suggested some concrete actions that...audience to use their useful energy and to choose institutions with allies engaged in training excellence to...action Each of the speakers in the panel had interesting words of experience and encouragement.  Dr. Sibby... training programs (like at the Keck Graduate Institute), funding for training (such as the NIH BEST award...

...end of your analysis process. You’ll still need to do statistical analysis to determine if there are any... differences of twofold or greater with good statistical significance (Taylor, 2017). With the right conditions...some general helpful principles (and I recommend Bustin, 2017 for a more in-depth review.)  Your amplicon...different efficacies DART-PCR No Raw Data Statistically defined log-linear phase   Table 2: ... them back before reporting the data!   Statistical analysis  Getting your RQ value isn’t the end... Yuan, 2006 has a good overview of different statistical analysis methods that pair well with slightly...step, remember we here at Addgene frown upon statistical laxness in all its forms. Reporting your data...

...outstanding questions you have, well before experimental crunch time. Don’t: Wait to ask questions and/or ...already read a paper or protocol. If you have questions while reading these materials (maybe a step in...then you’ve done previously) you can bring those questions to your training! All of these things will help...physically where relevant) and identify any gaps or questions you have, then bring them to your mentor. Pro...They can then plan accordingly in case you have questions or need a little assistance at some point during...yours is! For instance, some people like to ask questions throughout training, while others prefer to review...notes, process what they’ve just learned, and ask questions later – maybe even the next day. In the latter...

...vector systems. With the appropriate entry and destination vectors, one can use Gateway to clone a gene ...generated entry clone and the attR sites of the destination vector. This reaction is catalyzed by the LR ...containing the ccdB gene is excised from the destination vector.  Once the BP and/or LR reactions are ...select the positive clones. The entry clone and destination vector carry different antibiotic resistance ...gene is present in the donor vectors and the destination vectors prior to recombination, and it is exchanged...expression clone, it is important to choose the destination vector that best fits your experiment. This choice...doxycycline-inducible pLIX_403. The chosen attR destination vector will recombine with the attL-entry clone...

...nucleic acid detection is crucial for clinical diagnostics, genotyping, and biotechnological advancements...DETECTR demonstrate the power of CRISPR-based diagnostics. SHERLOCK- Specific High Sensitivity Enzymatic...lab demonstrated that SHERLOCK could reliably distinguish between Zika and a closely-related virus, Dengue... an array of applications including clinical diagnostics (e.g. pathogen or virus detection), therapeutics...purified from patient samples and can be read using a dipstick in under one hour. While the lab points out that... protocol serves as a platform for advancing diagnostics.     DETECTR- DNA Endonuclease Targeted CRISPR...lab demonstrated that DETECTR could accurately distinguish between two similar types of HPV, HPV16 and HPV18...

...first process by simply copying and pasting pieces from our existing script into the script block of the...several sequential computational tasks for which existing script(s) have been developed for a singular purpose...looking for was the ability to re-use or borrow existing code and control how the various pieces were connected...and optimize our pipeline. After reviewing and testing several platforms for engineering pipelines, we...resources. Now, we could iterate our development and testing cycle faster due to the ease of jumping back and...sequences on a 96-well plate basis, but for rapid testing and refinements, we specified a select small number...time and connect each subsequent process to the existing ones. Once our first process was working as expected...

...settings, and are fantastic tools for unbiased discovery. With these screens, the investigator generates a ...antibodies could thus be interesting drugs. Known drug targets  Another interesting approach is to design...CMM), Department of Medicine, Solna, Karolinska Institutet, in Sweden. It can be very time consuming to ...targeting each of the 1000 genes you’d like to investigate in your next CRISPR screen. Luckily, the Green...addressing a well-defined, hypothesis driven research question. Examples of custom CRISPR screens Differentially...particular, one slide of the presentation made a lasting impression on me. The slide showed a complex signaling... failing to upregulate the protein following stimulation would then be separated by sorting, and subsequently...

...samples of AAV for testing purposes. To address this, we recently released our serotype testing AAV, which are...peak fluorescence and resting fluorescence (achieved through lowering sensor resting fluorescence, while...AAVs include: EGFP-expressing AAV for serotype testing Calcium sensors and GABA sensors Optogenetics Recombinases...are distributed individually. See our Serotype Testing AAV inventory for the entire collection! We are... promoters and fluorophores for validating and testing purposes. We will continue to add more controls...responding variant. jGCaMP7b exhibits the brightest resting fluorescence and can be used for imaging small ...impacts neuronal circuits. We recently packaged a constitutive GABA sensor (see below), in addition to our ...

...Fig. 3: Restriction enzyme screening for resistivity to digestion, indicating targeted edits.   Interested...work, this is still a great option, especially if the options outlined above aren’t realistic. Check Out ...within a given genomic window, along with the estimated editing frequency of your sgRNA-Cas9 combo. Pro...be visualized by running your PCR on a gel. It’s still paramount that you sequence clones to verify that...homozygous clones, however, sequence confirmation is still needed to verify the correct sequence was inserted...base pair changes, but the edit must occur at an existing restriction enzyme recognition sequence or generate...solely for restriction enzyme sensitivity or resistivity, and thus be a marker for edited cells. As with...

...Lambert, 2019). These characteristics are important, but have less priority than stickiness. In my experience...has no strong affinity for other proteins, it may still block the function or activity of the tagged protein...proteins. Choose monomeric proteins and avoid a sticky situation Next to size, potential homodimerization...fluorescent proteins to oligomerize is known as “stickiness.” Since the fluorescent tag should operate as... per unit.  There are several ways to measure stickiness. In vitro, ultracentrifugation or gel filtration...oligomerization in cells. In addition to measuring stickiness, several researchers have also collected data...data on fluorescent protein characteristics such as the brightness, photostability, maturation time, and...

... models, and new Cre-lox based technologies are still being developed today. In this post, I will highlight...synthesis of alternative loxP sites to specify distinct patterns of recombination. In 2007, Joshua Sanes... robust single-cell labeling is necessary to distinguish individual cells. The Brainbow-1.0 construct ...OFP (orange), is expressed when Cre is absent; stochastic Cre excision results in one of three recombinations..., Sanes and Lichtman have observed from 90-160 distinct colors due to higher copy number. The name Brainbow...Although the fluorophores used are fluorescently distinct, their protein sequences are highly homologous... a majority of neurons, limiting the number of distinct colors that could be observed in a given area....

... field. Suggestions We provide as-you-type suggestions in different ways. Keyword suggestions inform the...catalog ID). Depositor suggestions provide shortcuts to matching PI labs and institutions. There are many more...websites, we built our new search engine atop Elasticsearch, a powerful open-source search engine that can...experience experts, our software team tailored Elasticsearch to support the different usage patterns the ...search bar, by ticking some boxes, by selecting a suggestion — how should these be supported and combined?...protocols for educational resources, and labs and institutions for depositors (see below). Even if the user...a search scoped to that category. Catalog ID suggestions provide shortcuts to specific catalog items as...

...synonymous mutations to distinguish it from the wild-type allele, they were able to distinguish between repair ...this blog was published, a bioRxiv preprint that questions the conclusion of inter-homologue recombination...mutations. But how close are we really, and what new questions does this paper bring up? We’ll sift through the... vs repair from the maternal MYBPC3 allele. Interestingly, they found that the embryos underwent repair...recessive gene. Ma et al. claim that CRISPR would still be helpful in increasing viable embryos, especially...surrounding CRISPR-based therapy is the level of testing needed to define whether off-target editing or ...be considered. It is important to consider the question of broader applicability of CRISPR germline editing...

...lab was very, very quiet. Every time I asked a question, I felt like I was yelling. I imagined 6 years...important decision. I’ll highlight some of these questions in the sections below. Mentoring relationships...all of the factors I touch on below, the first question to ask yourself is “which ones matter to me?”  ...lab is really like? Gather data of course. Ask questions. Prepare by going sleuthing on the lab website...for the interview with a long list of written questions for the PI and lab members.  Make coffee or beer... and whether or not it will work for you, ask questions like: Do lab members feel there are enough projects...what is best for you. Some of these important questions will be easy to ask the lab head, and some are...