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...they begin flowering. It is important to perform the transformation not long after flowering begins. If...RFP in the seed (Emami, Yee and Dinneny, 2013). Preparing plants for transformation Whatever plants you ...you can cut off the primary inflorescence (the flowering portion of the plant) near the base of the stem...al., 2006. Tips: Don’t plate seeds too densely during selection: this will increase background growth... (2002) Arabidopsis : a laboratory manual. Cold Spring Harbor Laboratory Press. Available at: https://...). Additional resources on the Addgene blog Engineering the plant genome using CRISPR/Cas9 Using pSiM24...pSiM24 for simplifying plant genetic engineering Using arabidopsis in the classroom Resources on Addgene.org...

...clicking “Add Plasmid to this Article” (left) and entering a plasmid name,plasmid type, and plasmid description...the plasmid names and descriptions first before entering the data. This step associates an Addgene ID with...plasmids all built with the same backbone and only differing in the gene/insert. Pooled libraries are normally... should click the "Enter Data" button to begin entering the specifics of your plasmids. Figure 5: Click... plasmid information. Enter plasmid information During the remainder of the process, you will enter plasmid.... You can complete these pages sequentially by entering the appropriate data and pressing "Save and Continue...species that the gene or insert sequence came from. Entering this information will populate a list of possible...

...customers. Details about our production protocols, titering methods, and quality control are described below... or through collaboration with viral vector manufacturing facilities, such as the University of Pennsylvania...are then aliquoted and stored at -80 °C. Titer Titering is either performed by Addgene or by the University...University of Pennsylvania Vector Core. In general, titering is performed by the facility that produced the...those achieved using the standard qPCR method.  Titering by the University of Pennsylvania Vector Core ...Purity Purity of AAV preparations is assayed by comparing the relative stoichiometric ratios of the viral...aliquoted, frozen, and stored at -80 °C. Titer All titering is performed on lentiviral preparations that have...

...system to partition themselves to each daughter cell during cell division. They don’t simply rely on chance...CBP), and a partition NTPase (Schumacher, 2012).  During cell division, CBPs bind to the centromere-like...replication that occurs for some types low copy plasmids during cell division simply does not allow for enough ...strategy has been successful in displacing, or “curing” the virulence plasmids from the bacterial pathogens... SR, Iredell JR (2017) Plasmid interference for curing antibiotic resistance plasmids in vivo. PLOS ONE...X, Wang D, Wang H, Feng E, Zhu L, Wang H (2012) Curing of Plasmid pXO1 from Bacillus anthracis Using Plasmid...22253811 Ni B, Du Z, Guo Z, Zhang Y, Yang R (2008) Curing of four different plasmids in Yersinia pestis using...

...appeal, instant sharing across multiple other sites), I created a new platform for sharing video abstracts...University and developer of wesharescience.org. Sharing your research with the world can be challenging...printed journal articles as the dominant tool for sharing ideas. This leads to everyday obstacles for many...www.WeShareScience.org. WeShareScience is a free platform for sharing short, 3 to 5 minute videos about research. Videos...oceanographer who has a blog and several videos on sharing your research through video. Liz Haswell and ... 525.7569 (2015): 306-307. 3. Spicer, Scott. "Exploring video abstracts in science journals: An overview...Scientific Impact Data Freedom: The Expansion of Data Sharing in Research Publications Resources at Addgene....

...the questions and preparing answers early in the day. Although I didn’t start answering until 5pm, frankly...well moderated. One of our original worries about sharing science advice on Reddit was that we’d be dragged... our AMA and Joanne’s preferred time to start answering questions. r/Science has pretty strict rules about...straight on through her designated two hours for answering questions. She was able to do this because we ...posted at 8:00 am and Joanne didn’t have to start answering until 5 pm). The ability to see the questions ... Tips: Online Social Networking for Scientists Sharing Your Lab Protocols: Using Apps to Save Time & Track...Experiments Additional Resources See How We're Sharing Science through Video with the Addgene Youtube ...

...culture has become overly confluent or too old. Ensuring that you are working with healthy, happy cells... the lentiviral stock needs to be titrated. If storing for less than a day, lentivirus can be kept at ...dry ice/ethanol bath or liquid nitrogen prior to storing. Know your target cells Labs tend to follow the...titer the virus (read this blog post for tips on titering your lentiviral prep!) and infect. While this ...consider is how the virus is being titered. Most titering approaches transduce into highly permissive cell...receptors, other important binding events take place during the course of infection. These binding events can...23589850. 9. Cronin, J., Zhang, X.Y., Reiser, J. “Altering the tropism of lentiviral vectors through pseudotyping...

...acceptance addresses were inspiring! We also got to hear more from them during a panel discussion with ...bestowed upon discovers of CRISPR/Cas9 genome engineering, Dr. Jennifer Doudna and Dr. Emmanuelle Charpentier...the presence of scientific heroines is always inspiring. I was especially excited to attend this event...enough to make up for the somewhat naïve questions. During the panel, an artist was sketching her interpretation...accelerate the launch of clinical applications. During the panel discussion, Dr. Mello took it upon himself... in the room. He offered the great analogy of comparing the CRISPR/Cas9 system to Norton Antivirus. Antivirus...

...of Plasmid Sharing: Interested in more statistics from Addgene's 10 years of plasmid sharing? Check out...our US counterparts are still sleeping). Plasmid sharing in Europe: Addgene’s request statistics Since 2004...Developmental Physiology at the University of Paris VII. During my years in research, I developed several new plasmids...is excited to be working at Addgene, because it brings scientists together from all over the world to ...discussing a new scientific discovery or invention, and sharing those ideas with others. This love of interacting... blog post Or look at our "10 Years of Plasmid Sharing" Infographic!  ...

...structure determination LEA proteins protect samples during cryo-EM grid freezing Engineered and evolved optimizations...2024.01.21.576499.    LEA proteins protect fragile samples during cryo-EM grid plunge freezing By Rachel Leeson Cryo-EM...can be damaged at the air-water interface (AWI) during the freezing process, resulting in sample loss....protective methods or detergents like CHAPSO, while requiring a lower concentration of sample — nearly ten times...-water interface protectant for fragile samples during cryo-EM grid plunge freezing. bioRxiv 2024.02.06.579238...prime editing systems for specific applications! Delivering prime editors (PEs) via engineered virus-like...off-target editing. Cargo size limits are also less stringent for eVLPs than other viral delivery methods, allowing...

...goals are (see monitoring task progress), and I know that I’ve blocked out time during my day according...transferable skills while working in the lab and by offering advice on areas where you might need some help... can spend accomplishing small tasks (like... preparing plasmids to send to Addgene). Following my advice...too many activities too close to one another. Monitoring task progress. For this, I always make sure I...-world example, let’s say I’ve blocked out time during my day to write a blog post, edit other blog posts...leave your phone in your bag on “Do Not Disturb” during the times you’ve blocked out for other activities...instance, while I may have found splitting cells boring, hanging out in the tissue culture room also gave...

... for monitoring RTK signaling.  Figure 4: Design of pYtag fluorescent biosensors for monitoring RTK activation...for investigating neuronal signaling and for engineering improved sensors for these and other targets....class of signaling receptors, but the options for measuring activity of specific RTKs are limited. In a new...colleagues describe pYtags, a modular system for monitoring specific RTK activation in vivo (Farahani et ...sensors. Using genome mining and structure-guided engineering of potassium binding proteins, they developed...and are a promising starting point for further engineering. Figure 5: Structure-guided design and genome...s41467-022-30685-x. Feel the heat with B-gTEMP  Measuring local temperature variations and heat transfer...

... recently showed that by delivering CRISPR components as RNPs and delivering two halves of a repair template... Precision Medicine, CRISPR, and Genome Engineering. Springer, Cham, 2017. 29-42. PubMed PMID: 29130152... used AAV gene editing to introduce naturally occurring cancer-causing mutations into the endogenous allele...homology arms with the modification of interest, ordering them from a gene synthesis company such as IDT...anaphase, and chromosome segregation in human cells harboring tumor-derived mutations in STAG2." PLoS genetics...Howes, Rob, and Christine Schofield. "Genome engineering using adeno-associated virus (AAV)." Chromosomal...

...of animals. There are many different naturally occurring strains of E. coli, some of which are deadly to...strain is a bit more convoluted due to researchers sharing and renaming samples throughout history. It was...cloning, routine cloning, cloning unstable DNA, preparing unmethylated DNA, and more. Many mutations that...Consequence dam DNA adenine methylase mutation (GATC) Preparing unmethylated DNA, important when trying to cut... dcm DNA cytosine methylase mutation (CCWGG) Preparing unmethylated DNA, important when trying to cut...relA1 Relaxed phenotype, mutation eliminating stringent factor Allows RNA synthesis in absence of protein...lacZΔM15] hsdR17(rK-mK+) JM110  Streptomycin  For storing plasmids that should not be Dam or Dcm methylated...

...technology since its initial applications for genome engineering in 2012.  Many of these advances have stemmed... fold induction at 31X. By using a slit pattern during blue light exposure (470nm), the researchers showed... C-terminal (residues 714-1368, C714) halves, rendering the Cas9 non-functional when split but regaining...strategy which had been engineered from naturally occurring photoactive proteins (i.e. CRY2 and Vivid) - Alexander... details, check out this comprehensive review comparing optogenetic CRISPR techniques (Matony et al., ...Built to Last. In: Methods in Molecular Biology. Springer US, pp 261–281 Manna D, Maji B, Gangopadhyay SA... Bratovič M, Charpentier E, Möglich A (2013) Engineering of temperature- and light-switchable Cas9 variants...

...those entering science communication (myself included) come from a scientific background and bring with... (and I hope I’m not alone in this sentiment). Entering the final semester of a neuroscience major, I ...first semester in which I had several projects nearing deadline (an animated video depicting nuclear fission...level of career satisfaction indeed.   For many venturing forth from tertiary study, the myriad trajectories... scientific research is, after all, one of the enduring goals of science communication.   The Owheo...improve the world around us has long been known; ensuring all have access to this power and the tools to...

... genome engineering, CRISPR is much more efficient at generating the desired mutant offspring, with 85%...adults mated to produce completely transgenic offspring. Although powerful, this technology is cumbersome... Advantages of using CRISPR for mouse genome engineering From the very start, generating mouse models ...microinjectionist, who has the strategically important job of delivering the payload! Last but not least, when using CRISPR...Marraffini LA, Zhang F (2013) Multiplex Genome Engineering Using CRISPR/Cas Systems. Science 339:819–823...Conditional Alleles by CRISPR/Cas-Mediated Genome Engineering. Cell 154:1370–1379 . https://doi.org/10.1016...Multiple Genes by CRISPR/Cas-Mediated Genome Engineering. Cell 153:910–918 . https://doi.org/10.1016/j.cell...

... body recognizes a drug as a foreign antigen, triggering a humoral immune response. ADAs can inactivate...inactivate the drug and increase drug clearance, lowering its efficacy and increasing the risk of an adverse ...on the immunogenicity of the drug product while ensuring the safety and the efficacy.  Better together ...determining the T-cell epitopes that will likely bring about unwanted immunogenicity. The 3-D models can...Monoclonal Antibodies in Macaca fascicularis: From Manufacturing to Preclinical Studies. In J. Bluemel, S. Korte...10.1634/theoncologist.2016-0061WHO. International monitoring of adverse reactions to drugs: adverse reaction...WHO Collaborating Centre for International Drug Monitoring, Uppsala, Sweden, 1992. Mattei, A. E., Gutierrez...

... visiting scientist is distinct from equipment sharing or a collaboration – it requires re-location of...training so that you yourself can become an expert and bring the knowledge back to your home lab. Alternatively...the conversation. Many researchers are open to sharing knowledge and establishing collaborations, so it...if they have specialized skills and equipment! Preparing for your visit   Design and communicate a research...mentor or anyone else available to directly help you during your visit. Reach out to your bench mentor (if ...and rights for anything that could be generated during the visit. If you don’t feel comfortable initiating...travels! Resources Resources on the Addgene blog Fostering communication in remote collaborations Transferable...

... Connie Cepko points out the limited choices in hiring graduate students and postdocs when starting out...counter-productive." He also emphasizes the point to “encourage sharing - in part by having projects ambitious enough that...position, start studying up before you begin the hiring process. Read Genevive Bjorn's "Ready, Set, Hire...Nature Jobs for some quick advice and resources for hiring research staff. And for a more indepth resource...experiments and grant writing. You have to enjoy the mentoring role.” She points out that new PIs may not recognize...student or postdoc may require different types of mentoring. Some mentees prefer lots of advice and micromanagement... interdisciplinary, with emphasis on genetic engineering, sequencing, and translation of results to companies...