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... of the acceptor fluorophore back to its ground state by emission of a photon with wavelength specific...FRET efficiency correlates with a change in the distance and/or orientation of the FRET pair. Since many... and serves as a small-scale ruler to measure distances that are too miniscule for conventional light ...contribute additional noise to the measurement. If the distance or orientation of the donor and acceptor proteins...undetectable FRET, well-characterized FRET reference standards can be used to validate FRET measurements and ...of the fluorescent protein (i.e. rearranging the start and end positions without changing the order of ...are not proteins) or would perturb the endogenous state that you want to measure (overexpressing a gene ...

..., or other. Bacterial Resistance Enter bacterial antibiotic resistance encoded in the plasmid that...considered large deposits and are exceptions to our standard workflow.  Table 1: Guidelines for the data fields... and will help a scientist outside your field understand if this plasmid will work for them. Species...yeast), S. pombe (fission yeast), A. thaliana (mustard weed), O. cuniculus (rabbit), synthetic, or other...Addgene should propagate your plasmids in: DH5α, NEB Stable, ccdB Survival, Pir1, or other. Whenever possible...possible, we propagate plasmids in the standard cloning strain DH5α. For plasmids with highly repetitive sequences...lentiviral, retroviral, and AAV plasmids, choose the NEB Stable strain. For plasmids containing the ccdB gene, ...

... are a great starting point: Test different lengths of the primer binding site, starting with a length...General principles pegRNA design If you’re just starting out with prime editing, you’ll need to design ...different lengths of reverse transcriptase template, starting with about 10–16 nucleotides. For longer templates...editing (Anzalone et al., 2019). Figure 2: Starting lengths for optimizing your pegRNA primer binding...BioRender.com. What to edit The sequence you choose to install can influence the success of your prime edit. If...template during DNA repair. Test multiple nick sites, starting with sites about 50 bp upstream and downstream...editors bound to sgRNAs only drive nicking (right). Start with nicking sgRNAs ~50 bp from the prime editing...

...them to begin understanding complex regulation of and by tRNAs and tRNA-derived RNAs. Stay tuned for big...lecture was presented by Nobel Laureate Jack Szostak. Dr. Szostak’s lab continues its work on the origin of...zone so many science and technology companies and start-ups choose to locate there. I made time to go into...traditional Chinese medicine. There were hundreds of stalls selling a myriad of items, only a small number ... Genome Research Institute (NHGRI) of the NIH, started off with the first keynote presentation. He talked... life. His lab has long hypothesized that life started with RNA and a simple cell membrane. His recent...they are making fairly remarkable progress on establishing a system for the chemical replication of RNA...

...without long distance communication and learning to work with people despite the distance improved my ...them “Leadership and Management skills”. The NPA states that Postdoctoral scholars should have the skills...management skills but a longer term and more substantial experience can be achieved by volunteering for...sharing my experience will show you how you can get started on your own development as a leader. Chairing a...styles Being an effective leader requires you to understand how to work with many different types of people...requirements of the new Board Members. In some instances I felt like I was going back to previous formats...beneficial for the organization as a whole. I came to understand that not only was I growing as a leader but the...

...However, there are instances where traditional base editors are preferred. For instance, if target nucleotides...availability than other methods. The PAM-to-edit distance can be over 30 base pairs for prime editing. Since...versatile and precise than base editing (in certain circumstances) Current base editors can perform any base-to-base...specific edit defined by the pegRNA. In cases when bystander editing is unacceptable, prime editors can be ...binding site, increase processivity, or improve thermostability. What came out on top? The Cas9 nickase fused... epegRNAs: Structured RNAs with improved stability Traditional sgRNAs are protected from cellular...al., 2022). PE7: An alternate approach to RNA stability This recent contribution by the Adamson lab also...

...about adeno-associated Viral Vectors (AAVs) before starting my graduate program at the University of Manitoba...and AAVs are non-enveloped viruses, so they are resistant to alcohol-based disinfectants. A 10% bleach solution...expression occurs quickly and results in long and stable expression (up to 9 months in mice [2]). Low Immune...tips that will help you think about how to get started with AAVs. Since my research is rooted in the CNS...Location, location, location! What do buying real estate and using AAVs have in common? The three most important... location, location! Just like this common real estate mantra, the choice of serotype (which determines... in the tissue/what cell type? In the CNS, for instance, there are marked differences among serotypes ...

...contributed by Laura Lee, a graduate student at Stanford University. Arabidopsis is a fantastic model organism...Agrobacterium tumeficans - a plant pathogenic species that stably integrates transfer DNA (tDNA) into the genomes... are also a variety of plant selection markers (basta, hygromycin, kananamycin, tunicamycin) (Nakagawa...plants. If your cultures have been incubated at stationary phase for too long, the cells in your culture...a colony from an LB plate. In my hands, a 5 mL starter culture inoculated from a colony on a plate will...density in 18-24 hours. I then add 0.5 mL of this starter culture to 100 mL fresh media, which is then ready...transformation in another 24 hours. The remaining starter culture can be used for colony PCR and glycerol...

...path.  A part of the career path story for most established grant professionals is learning to succeed in...often hear the term ‘grant writer’, but make no mistake, researching, winning, and effectively managing... with clear budget narratives, logic models, established partnerships and collaborations, and commitments...compliance with the grantor’s requirements as well as state and federal regulations. A grant professional’s ...no surprise that the U.S. Bureau of Labor and Statistics shows that employment of grant writers is expected... by 16% between 2010 and 2020. How did you get started in the grant profession? I asked Deborah Cook, ...on many different issues, and our program didn’t stay in one place within the city infrastructure. My ...

...guest blogger Sean Stacey! Sean D. Stacey is a Ph.D. student at East Tennessee State University. He works...This post was contributed by guest blogger Sean Stacey who recently attended both online and on site courses... online course had three aspects to it: videos starring Roberto Kolter, webinars taught by Peggy Cotter...were not too laborious, but helped build my understanding of how to generate a manuscript. At the end ...another email notifying me of my acceptance. I was ecstatic! I was going to become a serious scientific wordsmith...reinforced our newfound knowledge with practical understanding of how to apply it. The personal interaction...to get better. Well what does that mean? If you start a new technique, then type up the materials and ...

...discussing the review topic with your peers. Start reading! I started by reading other reviews because, as I...from a wide range of authors. Just start writing When I first started I thought I would read a bunch of... scientific review.” The second thing I did was stare in horror at the limited number of hits. So, here...outline the organization of the review Once you start reading, there will be a temptation to include every...normal day.” The way I broke this cycle was to just start writing. A word of advice: when reading, don’t feel... feel like you need to read every paper from start to finish. Find the information that’s relevant to ...results and contextualized them. No really, just start writing! Don’t worry about grammar or formatting...

...science majors, we do our best to understand the science. By understanding the hows and whys of our users...provides transparency to everyone involved about the status of each project. This creates accountability on...is our process for working on a new feature. We start with a story that describes the feature and what...collaborations between Addgene’s Product Team and the stakeholders (other Addgenies). Once we begin work on a story...closely with each other and with other teams. We start with writing demo details, step-by-step instructions...the Product Team who in turn collaborates with stakeholders to resolve the questions.  After the code is...from what I would have done. And finally the stakeholder accepts the new feature as done and we’re onto...

... Fulbright Visiting Scholar at the Pennsylvania State University. Imagine you are dealing with a defective...Transition mutation (C>T, G>A, A>G, and T>C) installation by base editors. Pathways that compete with ...They first created a defective chloramphenicol resistance gene (CamR) by introducing a point mutation (... adenine base editor would restore antibiotic resistance. To find such a protein, they created a mutant...systems. Thus, the Yang Lab at The Pennsylvania State University (PSU) is adapting and improving base ...developed plant adenine base editor (ABE) vectors to install A>G or T>C mutations in the plant genome. We constructed...     These binary vectors could be used to generate stable genome edited plants via Agrobacterium-mediated...

...after negative staining. Empty vector particles can be identified after negative staining and appear darker...generating a standard curve of known concentration. We regularly make and validate fresh plasmid standards. We...purposes in preclinical studies, it is important to understand what these values mean and how they can be compared...primer pair. Ct values vary based on the amount of starting material in the PCR because the sample must fall...fall within the linear region of the standard curve. In addition, high and low-concentration samples can..., which is a factor in AAV titration since the starting template is the purified viral vector solution...quantification by using the universal AAV Reference Standard Material (AAV SFM), which is an AAV sample that...

...on the cloning process, all constant regions, just the heavy chain constant regions or solely the Fc region...plasmid backbone containing all constant regions, just the heavy chain constant regions or solely the Fc region...great experimental plan that requires indirect staining of both targets in your specimen. You scour the...and heavy chains made up of variable regions and constant regions. Variable regions are unique to each antibody...antibody and convey specificity to the target while constant regions are identical for antibodies within certain...rat) are cloned into a backbone expressing the constant regions from an alternative isotype (purple, mouse...for multiplexing. In the case above, you can’t costain with your desired antibodies because the pair belong...

...in Table 2 is a standard and should always be included—the live/dead stain. This stain is in fact not ...in your analysis. Lastly, there are fixable dye stains that react with free amines on and inside the cells...antibody-fluorophores, fluorescent reporters, and/or dye stains used in your flow cytometry experiment are known...promote electrons within atoms to a higher energy state. This process is called excitation. After a short... period, the electrons revert to a lower energy state, whereby a photon is emitted. This step is called...laser but only "bright" for the blue laser. To understand why that is, let's look at Figure 2. The blue...markers with any fluorophore. Bleeding Before we start building your first panel, we'll talk about one ...

...guest blogger Sean Stacey! Sean D. Stacey is a Ph.D. student at East Tennessee State University. He works...This post was contributed by guest blogger Sean D. Stacey. Opinions on the apps discussed in the article ...great for lab life. These apps prevent you from constantly searching the interwebs for lab resources, and...computer. Lab Archives will help both you and your lab stay organized allowing you to upload anything from protocols... you. 2. Gibco Cell Culture by Gibco Whenever I start to learn a new technique, I try to look for a resource...for a mass spectrometry app. I digress. When I started learning proper cell culture technique, I tried...manual. Overall, it’s a great tool for someone starting cell culture work. 3. NEB Tools by New England...

...Nature report from researchers at Amgen, it was stated that only 11% of the academic research in the literature...due, at least in part, to insufficient and non-standard record keeping despite scientist’s best efforts...The familiar paper-based lab notebook is still a standard approach to record-keeping in many labs. In a ...functionalities that are hard to imagine on paper, such as instant search through all saved data and automated writing...writing of reports. To be able to understand the situation in depth and provide the solution that fits ...adoption within academic environments to better understand the issues labs are facing in terms of budget...to ELN adoption. This includes financial outlay, staff hours, troubleshooting, and the fact that long-term...

...undergrad working with cancer cell lines. As I started planning out my own experiments in graduate school... As a busy researcher, you’ve probably already started coming up with time management techniques of your...load yourself up with too many tasks. If you’re constantly switching between different projects, you might...make notes on as I progress. I’ve also recently started using Trello to manage larger, multi-person projects.... Knowing this, I hand write to-do lists at the start of each day to remind myself of the specific activities...where you’re pulled in a thousand directions at the start of the day, writing your to-do list the night before... each project and how useful each item is. For instance, if I don’t have draft blog post ready for publication...

...from the Institut Gustave Roussy. Professor Kroemer discussed the effects of starvation on cancer therapy...tumor-associated inflammation for therapeutic benefit ”as stated by the organizers. My overall impression from... that it is too fragmented and focused ... to understand cancer and defeat it we need to attack it from...treatment (Ref 1). As described in the presentation, starvation or the use of caloric restriction mimetics trigger...hepatocellular carcinoma, a cancer which can develop from a state of chronic hepatitis caused by hypernutrition and...consumption. Chronic inflammation is a dangerous state for the body to be in and usually self-limiting ...effects kick in trying to resolve the inflammatory state by inducing immunosuppressive measures, for example...