...wash buffer such as PBS or TBST. Stringent stripping The stringent stripping solution uses heat and detergent...your sample? Or what if something weird happened during your western and your results look… funky? Do you...along with the antibodies, especially with more stringent stripping methods. If you are planning to detect...that section of the blot unusable for reprobing. Preparing your western blot Run your gel and membrane transfer...primary and secondary antibodies from your membrane, clearing the way for the second blot. There are multiple...with a mild stripping solution and move onto a stringent solution if the mild is not effective. You may...perhaps using longer incubation times, or try the stringent solution if you started with the mild one. Reprobe...

... steps, until its connection with DNA is broken during cross-link reversal (not shown in diagram). Cross-links...sequence targeted in the experiment. Figure 2: Comparing CAPTURE and CAPTURE 2.0.     Original CAPTURE...) one or more gRNAs Two separate lentiviruses delivering 1) a BioTAP-tagged dCas9 and an eGFP tag, and...detecting chromatin interactions opens the door for answering more questions about chromatin conformations. ...‘social networks’ of 157 super enhancers.  3. Measuring temporal changes in chromatin interactions A third...in chromatin conformation, like those that occur during development and cellular differentiation. Using...the Xu Lab tracked enhancer-promoter interactions during erythroid differentiation. CAPTURE 2.0 captures...

...packaged inside the vectors during production. These impurities cannot be removed during the purification step...recombinase-independent recombination during plasmid growth in bacteria or during viral production. This recombination... control assays for AAVs. These steps include measuring viral titer and assessing purity. But Addgene ...wrong plasmid? What if bacteria snuck into the prep during the production process? These questions are especially...materials. Addgene is going the extra mile and pioneering the systematic use of this new QC assay to guarantee...Evidence for Encapsidation of Prokaryotic Sequences during Recombinant Adeno-Associated Virus Production and...

...of choice is AAV, with nearly every experiment requiring AAV. Before joining my lab, I had never worked... a heated stir plate to dissolve and requires monitoring so that the solution doesn’t get too hot because.... Rather than investing all of this effort in preparing reagents, I choose to only harvest the cell pellet...layered and then the viral suspension generated during AAV production is overlaid. Following ultracentrifugation...first purification. This helped me get a feel for layering the iodixanol solutions and let me double-check...make one or two small aliquots to use for qPCR titering. This helps avoid multiple freeze/thaws of your...of virus. AAV titration by qPCR Overview: AAV titering by qPCR quantifies the number of genome-containing...

...path began to take shape, was when I started volunteering. I wasn’t sure yet what a career in science ...volunteered at a place that made me happy. I started volunteering in the “Dinosphere” almost every week. I gained...dinosaurs, and met wonderful people. When I started volunteering, I was worried about becoming one of those angry...t sure what I wanted to do. A few weeks into volunteering, I was talking to a little girl about fossils...Beyond the outreach experience I gained from volunteering, it gave me a place to go once a week to get...before founding CINSO. Now, the network I built during graduate school is my informal advisory board. ...to share valuable life lessons they have learned during their scientific training and wrote about them ...

...the palm of your hand. Although white light is entering your hand, only orange/red light can be seen exiting...optical section is achieved that boosts contrast by filtering out emission light from fluorophores higher in...as spinning disk confocal. However, due to the scattering nature of biological tissue, imaging more than...parallelization (7) have allowed for real-time monitoring of neuronal activity across substantial 3D volumes... allow for multi-view imaging can image thick scattering samples in their entirety, something that is ...excitation light which can lead to photobleaching when acquiring multiple images over a large axial range. 2-photon...overcome this (8). The recent development of tissue clearing techniques (9) now allows researchers to image...

...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...

...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...

...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...

...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!  ...

... 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...

...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...

... 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...

...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...

...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...

...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...

...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...

... 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...

... 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...

... 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...

... 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...

...world of academic research can be quite daunting. During my first year of college, I knew I wanted to try... four years after I started college, I will be entering a PhD program to continue my scientific endeavors...met graduate students who enjoy the thrill of acquiring a piece of knowledge before anyone else in the...neurodegenerative disease because they had close relatives suffering from similar diseases. Others were inspired by...biofuel production. There’s a lot out there worth exploring! Being excited about the science allows you to...proactive Rather than applying only to posted job offerings, the best research experiences typically come ... provided me with much-needed emotional support during my journey. When you are hunting for labs to join...

... neurons using GCaMP7, measuring calcium signaling related fluorescence during morphine administration... neurons at specific firing frequencies. She hopes to identify how in vivo firing frequency of PV neurons...researchers attend a conference of their choice while honoring the late Michael Davidson and Roger Tsien who ...iGluSnFR3 (left). GluSnFR3 in pMinDisplay (PDGFR anchoring domain), in electroporated in rat hippocampal ...modulate protein activity and have important roles during oxidative stress. To monitor this activity, Pedre...

...allow authors to avoid many of these issues, by sharing their work publicly and immediately once the manuscript...emerging SARS-CoV-2 virus quickly and openly. By sharing results faster and more broadly, scientists can...evidence of their productivity that can be shared with hiring and review committees, as opposed to the opaque.... Preprints at Addgene Preprints are all about sharing scientific knowledge faster and more openly - which...open-science organizations, we support scientists in sharing their results and materials with the broader community...in a preprint provides all the same benefits as sharing those plasmids alongside a journal article, but...He also posted two preprints of his own research during his PhD studies.  References and Additional Resources...

...take a look at a few of these methods below. Engineering gut microbes in situ While the ability to monitor... University developed a method for genetically altering gut microbiome members in their native habitat...reporter gene. The hairpin blocks translation by sequestering the RBS, but binding of the “trigger RNA” (in... Counting cell division in the gut microbiota Monitoring microbial population dynamics in the gut can ...one bright particle that segregates into one cell during cell division. To achieve the agglomeration of ...References Leonard, Sean P., et al. "Genetic engineering of bee gut microbiome bacteria with a toolkit...PMC4674677. Ronda, Carlotta, et al. "Metagenomic engineering of the mammalian gut microbiome in situ." Nature...

... genetic engineering, with the ultimate objective of genome design.” It’s genetic engineering on a genomic...toolbox needed for anyone to begin considering genetic engineering projects in a very big way. In one ...scale could be created. Someone could take on an engineering project including hundreds of genes and regulation...complete toolbox for taking on very large genetic engineering projects. Addgene: How easy is it to do this ...animals make. That will be an outcome of genetic engineering.   Thank you to Chris Voigt for speaking with...

... Cold Spring Harbor Protocols. 2012 Jun 1;2012(6):pdb-top069609. Palmer AE, Tsien RY. Measuring calcium...concentrations. This will reduce noise and ensure you are measuring the events-of-interest. Sensors suitable for ER...reporters use a single fluorescent protein (FP), requiring only a simple measurement. However, single wavelength...sites per sensor (to decrease unnecessary calcium buffering and to increase sensitivity), and is a great FRET...expression and integration within the target cells, ensuring more consistent and reproducible results in calcium...subtypes for efficient expression and downstream monitoring. This targeted approach allows scientists to ...

...In order to understand the complex wiring of the brain and the underpinnings of neurological disease,...manipulating cell activity, especially neuronal firing. What is chemogenetics? Chemogenetics relies on...looking at immediate effects of specific neuron firing are better equipped using fast-acting optogenetic...receptors can be used to alter excitability by lowering or raising the threshold for signaling. The two...for neuronal signaling and causes neuronal burst firing. Other DREADDs, such as the KORD receptor, increase...The Power of the Lock, the Weakness of the Key. Favoring the Pursuit of Specific Conditions Rather than...Carpenter JC, Snowball A, Knauss S, von Schimmelmann M, During MJ, Lignani G, Schorge S, Young D, Kullmann DM,...

... while engineering viruses Acr genes can be used as positive selectable markers in engineering viruses...Acr proteins can also be used to prevent dCas9 tethering to DNA. Since these early anti-CRISPR studies,...dimerize which blocks the PAM recognition site (Harrington et al., 2017). Inhibit crRNA loadingExample: ...domain of Cas9 and prevents target DNA cleavage (Harrington et al., 2017). Figure 1: Anti-CRISPR proteins...436–439 . https://doi.org/10.1038/nature22377 Harrington LB, Doxzen KW, Ma E, Liu J-J, Knott GJ, Edraki... EJ, Maxwell KL, Davidson AR (2016) Naturally Occurring Off-Switches for CRISPR-Cas9. Cell 167:1829-1838...

...amount of time. Though I now spend far less time answering help emails and phone calls, I feel compelled ...can be a particularly good resource if you are considering using a reagent for “off-label” purposes. When... than you might think. The Addgene scientists answering the phone are smart, but they cannot (and should..., but many bio companies have real scientists answering their technical support questions (besides Addgene...Addgene, I know both Abcam and NEB have Ph.D.s answering emails and phones). We can’t be experts on every...-up when things get crazy. Take home message Answering customer service questions is a delicate balance...Once You Recieve Them Get Help with the Addgene Ordering Process ...

..., the vector may undergo genomic rearrangements during replication. To avoid genomic rearrangements, try...store freshly harvested virus at 4 °C instead of storing it briefly at -80 °C. If troubleshooting, the safest...increased by concentrating a viral stock. Different titering methods can reveal differences in physical versus...result of plasmid integration via recombination during transfection, and does not necessarily indicate... infect the packaging cells. Another method of ensuring viral production is to perform PCR against components...adoptive immunotherapy." Acute Leukemias VIII. Springer Berlin Heidelberg, 2001. 291-306. PubMed PMID:...Yezzi, and Barbara R. Young. "Recombination events during integration of transfected DNA into normal human...

...CRY2, although some studies have shown that this clustering only happens under certain conditions. With...drawback of this method is the stray excitation of neighboring neurons in opsin dense regions. To overcome this...able to demonstrate that while illumination of neighboring cells evokes action potentials (APs) in patched...this operon can serve as a living biosensor for measuring caffeine content in samples. The Barrick lab and...concentrations in several beverages could be estimated by measuring growth of the ΔguaB pDCAF3 strain. Find the decaffeination...placesTagRFP-T and Dronpa in close proximity. By monitoring changes in TagRFP-T fluorescence intensity at...the authors tracked protein activity while also capturing information about spatial and temporal organization...

...guide sequence for your genome engineering experiment. With CRISPR entering the clinic for correction of...structure-guided engineering of the PAM-interacting domain of Cas9. After multiple rounds of engineering, they developed...PAM right where you need it, the odds of this occurring are not in your favor. Thus, there is a need to...coverage in the human genome. SpRY Cas9 has PAM sites covering the majority of the genome! Want to get your...Requirement and Expanding Beyond Cas9 xCas9: Engineering a CRISPR Variant with PAM Flexibility History...

...population. Outbred strains of mice are useful for comparing dominant and recessive traits and their impact...or treatments, but are not useful for genetic engineering. Examples of outbred strains include Swiss Webster...generating knockin mice but because CRISPR/Cas9 engineering favors DNA repair via non-homologous end joining...have errors introduced to the nucleotide sequence during the DNA repair process. The addition of small molecules...to transgenic mice, knockout mice are engineered during the embryonic stem cell stage using one of several...blog posts about using Cre-lox Read our genome engineering blog posts Resources on Addgene.org Find more...more about Cre-lox Read our Genome Engineering Guide Learn more in our CRIPSR guide ...

... important consideration when preparing the implant itself, which brings me to the second key point; the...cable to the ferrule implant must be maintained during the entire protocol, otherwise optical stimulation...it to a patch cable connected to the laser and measuring its output with a light meter before and after...that one laser can stimulate only a single animal during the course of an experiment. There are a number..., 6]. These have the obvious advantage of not requiring patch cable connection to the laser, which affords...science writing. Derek is also a pianist and an aspiring composer. View his blog drsimonsaysscience.org...

...of Revolution Bioengineering.  Last year we started a company. Revolution Bioengineering is two responsible...so hard that we upended our lives and took this daring risk? What moment changed our whole outlook on ... We both had an applied science bent, so the engineering aspect of this job appealed to us. However, since...thinking about how we could bypass the establishment hiring system altogether. We had a lot of ideas about ...molded the whole thing into a marketable product. During this process, we had an idea that survived the ...outside of Washington D.C. and has always enjoyed exploring the outdoors and experiencing nature first-hand...

...2023). Uncovering the functional diversity of rare CRISPR-Cas systems with deep terascale clustering. Science...interest in bacterial and archaeal immune systems. During this time, scientists discovered many related immune...keep increasing, as efforts using large-scale clustering and discovery algorithms find new candidates ... L. A., & Zhang, F. (2013). Multiplex genome engineering using CRISPR/CAS systems. Science, 339(6121),...1027. https://doi.org/10.1126/science.aaq0180 Harrington, L. B., Burstein, D., Chen, J. S., Paez-Espino..., K. V. (2021). Type III CRISPR-CAS systems: deciphering the most complex prokaryotic immune system. Biochemistry...(2015). Co-transcriptional DNA and RNA Cleavage during Type III CRISPR-Cas Immunity. Cell, 161(5), 1164...

...experiments simultaneously measuring the activities of CaMKII and RhoA during induction of structural plasticity...type. To this end, they constructed AAV plasmids bearing one of the distal-less homeobox 5 and 6 (Dlx5/6...“caged” by fusion to specific domains thereby rendering them inactive in the cytoplasm. The authors use... Segment on PhoCl LSSmCherry1 & RDSmCherry1: Engineering and directed evolution of new RFP variants The... length with TZAP Multiplexible plant genome engineering toolkit The Voytas Lab at the University of Minnesota...used for either TALEN or CRISPR -based genome engineering across a variety of plant species. This toolkit...variety of promoters for versatile and robust engineering. These components are provided in Golden Gate...

... first identified in the late 1960s during research at hot springs in Yellowstone National Park.  In addition...Using these reagents and a series of heating (denaturing) and cooling (annealing) steps Taq polymerase... common for dNTPs and other reagents to run low during the final cycles which would slow or stop PCR amplification... as real time PCR is a PCR technique used for measuring a starting DNA concentration using PCR. qPCR requires...this fluorescent dye, the concentration of the DNA during the PCR reaction cycles is continuously detected... Variations Quantifying low abundance targets Monitoring subtle changes in target DNA levels Multiplex...

...significant activity on some mismatch-containing pairings. During Golden Gate assembly, ligation of mismatched... assemblies that enable biosynthetic pathway engineering and optimization. However, current best practices...redesign was guided by both advances in the re-engineering of the original BsaI-HF® Type IIS restriction...efficiency and identification of mismatch-prone pairings. Using these 4 bp overhang observations, accurate...and a Development Group Leader overseeing a team bringing new products to market, New England Biolabs, Inc...comprehensive profiling of T4 DNA ligase fidelity and bias during DNA end-joining." Nucleic acids research (2018)...

...you to our depositors for sharing your science with us! Corralling and Cornering the Goldilocks Antibody...editing and multiplex prime editing (requiring minimal payloads!) Uncovering Ancestral Bias in CRISPR Research...antibodies for use in mice and human brain IHC and sharing the data on the mAb3D atlas website.  The iGluSnFR3...Beroukhim labs to write a post on their paper uncovering ancestral bias in CRISPR guide library design...

...becoming an ever more ubiquitous tool for genome engineering, and studying its structure can help us understand...nucleotides 1-10 of the guide RNA, prearranged for base pairing with a complementary DNA strand. These 10 nucleotides...Figure 3 crystal structure, reflecting the less stringent recognition mechanism that can tolerate mismatches...mismatches in the next 8-10 bases after perfect pairing in the seed sequence.   Figure 6: Electron...stabilizing the active conformation of the HNH domain, ensuring all binding partners are in place before cleavage...still being uncovered, and different techniques all bring their own advantages—and blind spots—to these questions...better understand how to manipulate it. Happy CRISPRing!   Molecular graphics and analyses performed with...

...Reaching one million plasmids shared might have you wondering how many plasmids leave Addgene headquarters every...Joanne Kamens, Addgene’s executive director, said during our celebratory lunch, it’s hard to picture one... September 13, 2018 This massive scale reagent sharing led us to calculate how much time we have saved...would have saved 5 million hours through reagent sharing. How much is 5 million hours? A colossal 547 years...plasmids to the repository and for the scientific sharing community (check out the infographic below for ...the past month, we’ve enjoyed seeing scientists sharing their contributions on social media as well the...

...the host cell. Some of these envelope-receptor pairings are broad, allowing delivery into many cell types...allows these viruses to infect neurons. Besides altering the host tropism of your virus, there are many...reasons when you might pseudotype your virus:  Lowering cytotoxicity. Different envelope proteins have... up less of the virus and is less affected.  Altering sensitivity to serum. Different pseudotyped viruses...the TVA receptor in neurons using AAV and then delivering rabies dG pseudotyped with EnvA. This allows ...v12050513 Cronin J, Zhang X-Y, Reiser J (2005) Altering the Tropism of Lentiviral Vectors through Pseudotyping...Quick Guide to All Things Lentivirus Get tips for titering your lentiviral preps Resources on Addgene.org...

...pEcCas/pEcgRNA system. This system works well for engineering single genome edits but the double strand breaks...Cas9, a temperature-sensitive replicon for self-curing, an arabinose inducible lambda-red gene, and an... E. coli B and W strains. The optimized plasmid curing method was much simpler and took only ~32 hours...sites edited. We optimized the strategy for plasmid curing, providing users with a complete editing process...interrupt genes (or gene clusters) in metabolic engineering. So far, we’ve used the MUCICAT system in two...compared to the industrial strain. In our metabolic engineering case study of N-acetylglucosamine biosynthesis... systems to choose from. Ready for metabolic engineering with improved cargo dedicated to gene (cluster...