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...Frequently asked questions Now let's get into some of the nitty gritty and answer the questions those of us...reagents as outlined by your institution or country. Have any other questions? Feel free to post them in..., and more. Not surprisingly, we receive many questions from scientists all over the world looking for...answers to our most frequently asked lentiviral questions. This useful resource from Addgene Depositor ...quickly check that a construct is functional before investing time and resources into making a stable cell line...is useful in gene therapy applications where a distinct subset of the cellular population needs to be ...aspects of lentiviral transduction along with suggestions on how to lower the risk. Biosafety should always...

...yet powerful way to tag cellular proteins and investigate protein localization, interaction, and expression...reversible photoactivation - and have enabled sophisticated imaging techniques.     Irreversible photoactivation...victoria) GFP (wtGFP) by mutating Threonine 203 to Histidine (T203H) (1). wtGFP normally contains a mixed population...can occur multiple times by activating with two distinct wavelengths of light (see Table 2). The best known...usually preferred for studying single protein characteristics, as the monomeric state is less likely to interfere...super-resolution imaging usually have the following characteristics-high contrast ratios, bright fluorescence, ...conventional FPs for super-resolution imaging, they still do not emit the large number of photons achievable...

...to the other six mentoring questions in future posts. If you have a question about mentoring that you want...Cepko to learn about her mentoring style. Top 7 questions scientists ask me about mentoring In this blog... series, I plan to provide answers to the top questions scientists ask me about mentoring. Here they are...’ – inspire you.” “Also a mentor can give you realistic expectations. (Don't think you can finish that... is a sounding board--a good mentor asks more questions than answers them.” A good mentor does all of...lighthearted, the article does highlight key characteristics of a good mentor: Yoda image courtesy...Check Out Angela Depace's Talk on "Precision and Plasticity in Mentoring" A recent post on the blog “Tenure...

.... Kutubuddin Molla, investigator at ICAR-Central National Rice Research Institute. When it comes to genome...Kutubuddin Molla is an investigator at the ICAR-Central National Rice Research Institute in India. He specializes...soccer ball, TnpB is a baseball — much smaller, but still powerful. Our lab recently harnessed TnpB from Deinococcus... and guide design rules.   Step 3: Vector Digestion Digest either pK-TnpB1 or pK-TnpB2 with BsaI....plasmid is now ready for Agrobacterium-mediated, biolistic, or PEG-mediated transformation into plant cells... genome editing vectors. If you have specific questions, feel free to contact the Molla Lab (X account...https://doi.org/10.1111/pbi.14416  Maher, M. F., Nasti, R. A., Vollbrecht, M., Starker, C. G., Clark, M...

...organization also known as external collaborations. Interestingly, the ability to work with others outside one...important as more complex approaches to scientific questions become popular. There are only so many methods... in Strategic Partnership Management, a very interesting and exciting part of my work. However, this is...collaborations. During my graduate studies at Karolinska Institutet I started working with other groups. First within...minutes. Get to know your collaborators from other institutions personally on conference calls and make sure...opportunities so just pick the one that you feel enthusiastic about and get started. You never know where ...collaborating with your neighboring lab, you've met interesting people at conferences and you have successfully...

...publication at Addgene. Walton RT, Christie KA, Whittaker MN, Kleinstiver BP (2020) Unconstrained genome ...upstream of either NGA or NGC PAM sequences (Kleinstiver et al. 2015). From these screens, they identified...off-target activity (Hu et al., 2018).  In 2020, the Kleinstiver lab reported the development of near-PAMless .... Furthermore, non-SpCas9's may have other characteristics that make them more useful than SpCas9. For... in target genes resulting in the formation of “sticky ends” rather than the blunt ends created by Cas9...which makes it easier to target AT-rich genomes (Kleinstiver et al., 2016). Type VI CRISPR systems, which ... plasmids from this publication at Addgene. Kleinstiver BP, Prew MS, Tsai SQ, Topkar VV, Nguyen NT, Zheng...

...Resistance can be intrinsic, meaning a basic characteristic that naturally occurs in the bacterium, and...but has become resistant somehow overtime. One fantastic example of intrinsic resistance would be Mycoplasma... very skilled at the delicate art of survival. Existing virtually ubiquitously in the environment, there...infecting bacteria can take 24 hours or more, and testing for resistance often takes an additional 18-24 ...available. For routine infections, like strep throat, testing for antibiotic resistance isn’t commonly performed...infections is so rare. When antibiotic resistance testing is requested clinically, this involves growing ... antibiotic resistance?  The problem with this question is, unless an antibiotic targets an essential,...

...hybrid guide to be effective. Which bases can be substituted? A guide RNA has many parts. The ~20 base “spacer...dramatically reduce CRISPR efficiency, and DNA substitutions don’t fare much better: they largely abolished...recognizes the 2’-OH of RNA in this region, and DNA substitutions dramatically reduced cleavage (Kim et al., 2020...some parts of the scaffold sequence could be substituted without significantly reducing genome editing...scaffold sequences have different tolerances for substitution of DNA bases. Created with BioRender.com. ...matches, the complex is stable enough that the DNA substitution doesn’t disrupt it. Combined with the reduced...access these kinds of tools. Location of DNA substitution Similar on-target efficacy Reduced off-target...

...025-09021-y  Walton, R. T., Christie, K. A., Whittaker, M. N., & Kleinstiver, B. P. (2020). Unconstrained...whirlwind of developing cell and mouse model systems, testing base editing variants, and assessing the safety... mutation. To tackle this problem, Benjamin Kleinstiver’s lab engineered new variants of S. pyogenes Cas9...came out on top. Narrowing PAM preferences The Kleinstiver lab also worked on the opposite project: engineering...tested mutations produced efficient editors, the Kleinstiver set performed best when paired with the V106W... in different patients. While these gRNAs would still need to be optimized and assessed for off-target... J., Butcher, R. M., Pacesa, M., Smith, B. K., Christie, K. A., Ha, L. L., Meis, R. J., Clark, A. B., ...

..., an individual experiences progressive muscle wasting leading to death at around 30 years of age. Despite...the amount of research conducted on DMD, there is still no good treatment. The dystrophin gene is very large...approach. Since shorter forms of dystrophin can still be functional, exon skipping is a good option for...non-homologous end joining (NHEJ) pathway. It’s estimated that very little dystrophin correction (about ...could make a huge difference in DMD, and it’s estimated that exon skipping therapies would be applicable...experiment, they found the exon-skipped transcript constituted 59% of total dystrophin mRNA, similar to the ...CRISPR therapy closer to the clinic, much work is still needed. First, CRISPR delivery must be optimized...

...to non-jargon in the context of disease testing, it can still be considered jargon in most other uses....situation rather quickly!  The question   The first, and most important question, you should ask for any science...ll see throughout the posts and introduce *the* question underlying all communications efforts.  Scicomm...scientific or science communication. A Tweet is still considered scientific communication if the language...An audience can be defined by any number of characteristics. Common ones include age range, familiarity...patients or caregivers affected by the disease in question who are involved in online patient/caregiver communities...

... preparations undergo rigorous quality control testing at Addgene meaning they come ready made to accelerate...researchers lamenting the viral production process while still ordering viral vectors. Virus production can be ...interested in faster by producing, titering, and testing viral preps for them. At the same time, we’re developing...base for the processes of viral production and testing so that researchers can use our educational resources...researcher Connie Cepko has been helping us with AAV testing and recently had this to say about the service:...suggest additional plasmids with the Viral Service Suggestion Form. Lentivirus As some of the most popular ...well as other expression data. If you have any questions about the Viral Service or any of the materials...

...majority of research institutes.  Pro tip! In most academic and nonprofit institutes, the Tech Transfer ...our process easier for others.  If you’ve still got questions about our MTA process, you can check out ...commonly used ones for transfers between academic institutions.  What is the UBMTA, you ask? Well, organizations...people think our MTAs are different than other institutes. But they’re not! The vast majority of our plasmids...’s important to note that it’s the depositing institute that decides the terms of distribution for their...of coordinating signatures from two different institutes. Our online portal automates a lot of the process...

...action and could potentially stimulate the development of useful diagnostic methods and improved therapies...for PZA-resistant Mycobacteria tuberculosis. Investigation of multispecies biofilm formed by Pseudomonas...compounds on these proteins will be tested. Investigating changes in neuronal activity after ischemic ...ischemic brain injury Dr. Gohar Tsakanova, Institute of Molecular Biology of the NationalAcademy of Sciences ...Armenia Gohar Tsakanova is a Senior Researcher who investigates the molecular and cellular etiopathomechanisms...non-linear microscopy methods will be used to stimulate and manipulate neural populations in order to ... Julio Ernesto Valdivia Silva is a master of interesting combinations: biomedicine and astrobiology. During...

...adjust our estimates based on the journal that published the articles.  That is, we re-estimate the citations...correlation. But even with that caveat, it is still suggestive that we see such a big difference in citations...than those without Addgene deposits? Even more interestingly, could we tell if depositing a plasmid with ... This is also true for other journals, so our estimates now take into account any differences in citations... That papers with plasmids deposited at Addgene still accrue on average 2 to 3 times as many citations...step. There are lots of other factors that could still be controlled for. As an example, plasmid-related...

...Hernández-Sánchez is a postdoctoral researcher at Instituto de Investigación Biomédica de Salamanca (IBSAL) and one...the competitiveness of cutting-edge research, is still one of the most valuable features of modern science...researchers, such as postdocs and principal investigators. They also come from many different scientific...-and-error. A recommendation for the correct statistical test can save a retraction. Today, collaborations... favors from becoming starting points for long-lasting partnerships Join us! We are a community of researchers...about us and what we do in our Frequently Asked Questions. We also invite you to participate in the 1st ...16:00 CEST. This symposium aims to answer all questions you may have about Crowdfight and we will have...

...experiments.” - Alex Chavez, researcher at the Wyss Institute for Biologically Inspired Engineering and member...alleviate this frustration by producing, titering, testing, and providing lentiviral preps of select lentiviral.... For example, we’ve teamed up with the Broad Institute to provide lentiviruses for delivery of SpCas9...they require extensive background work from the requesting scientist for their proper use; after receiving...already expressing Cas9. As John Doench (Broad Institute and member of the Addgene Advisory Board) says...about using our viral services. Please send any questions or comments to [email protected]. Finally we’d ...and, in particular, the Church lab at the Wyss Institute for Biologically Inspired Engineering and the ...

...maintain fidelity with the stimulus. For example, if you want to stimulate at a fast frequency without...hardware types, construction, and testing used for delivering your light stimulus. This can make or break your...that induce conformational changes with light stimulation to either activate or inhibit a specific receptor...cryptochrome 2 (CRY2) (Zhang & Cui, 2015). When stimulated with light, the endogenous proteins activate ...slower kinetics can be used for lower frequency stimulation. GPCR signaling GPCR signaling is a complicated...be very different responses to long vs. short stimulation times or frequencies (Zhang & Cui, 2015). You...Bendifallah, I., Zhou, F., Zeitzschel, N., Ahlbeck, J., Augustin, S., Sauter, K., Papagiakoumou, E., Gottschalk...

...someone asks a question about something they did not understand about your presentation, question whether other...beneficial to pose these why questions to yourself, but ultimately there is no substitute for feedback from others...research proposal is the perfect fit to their institutional focus and that you're better equipped than anyone...audience think or act.   To address that 'why' question effectively, you need to make your audience the...communication should prepare you to ask and answer these questions for a diverse set of audiences.  Every scientist...opportunities to practice communicating.  Some fantastic K12 education initiatives like BiteScis (https...

... social media conversations, conferences, and questions our scientific support staff receives – in fact...and share in-depth answers to frequently asked questions. We also want to tell you about things you didn...articles answering all of our frequently asked questions!    Identify needed content Let’s take a look...successfully start using them at the bench, along with questions we thought they might have as they go. The guide...things and another for asking specific science questions. It’s not uncommon to see requests for fact checks...of our content already comes from you and the questions you ask Addgene and your research community! We...engaging and informative resources that answer questions our depositors and customers are asking. So how...