...screen using PCR and a restriction enzyme digest.   Fig. 3: Restriction enzyme screening for resistivity...of your experiment for a screening strategy. Restriction enzyme screening is sensitive to single base ...changes, but the edit must occur at an existing restriction enzyme recognition sequence or generate a new... but your edit doesn’t affect any endogenous restriction enzyme sites? Never fear! You can introduce a...silent mutation should be designed solely for restriction enzyme sensitivity or resistivity, and thus be...

...forward to in 2023 and beyond. Last year, we distributed 165,000 plasmids, including 27,000 CRISPR plasmids...and general-use antibodies, is available in both trial- and full-size, with assay-supported recommended...our website saw 2.2 million visitors from 231 countries. Guides and protocols were our most popular content... million page views from visitors in over 200 countries. We, like everyone around the world, are learning...research community through sharing science and distributing high-quality materials and information. As a...

...When performing restriction digests on plasmids at Addgene, we sometimes observe something odd in our...various multimeric forms (e.g. concatemers, dimers, trimers, tetramers). Since then, multimerization has been...themselves cannot be used to detect multimers, since restriction sites in multimers usually repeat in tandem along...DNA without changing the size. As a result, a restriction digest of multimers will produce the same fragment...sample, you could try linearizing the plasmid via restriction digestion and then run the linearized plasmid...eBookAddgene's Molecular Biology ReferenceAddgene's Restriction Digest Protocol More resources on the Addgene...

...This post was contributed by guest bloggers and IMP organizers Rosario F. Godino, Rocío López-Diego, ...youth, our future, around the world.   In many countries of the world, professional internationalization.... Brain drain is a serious problem in several countries, however programs like IMP, bring closer than ...from talented professionals to their original countries to help build, together with the universities,...pre-program self-assesment. Achievements directly attributable to participation in the program included improvements... will also initiate mentoring pilots in other countries. In 2016, we expect the program to comprise over...

...nights, game nights, and trips to eat out together. “We also have a lab day trip each year and as we do ... the lab receives send-off via a gift, card, and trip to the pub. Dr. Ellis adds “we probably don’t celebrate...lot of yeast research, a few of those have been trips to breweries who happily exchange our expertise ...company outside of the lab by sharing meals, going on trips, and going to New Hampshire in the summer for the...

...through a distributor?  Yes! Packaged on Request service will be available through our distributor partners...of AAV plasmids will have technical or legal restrictions that make them ineligible for use in this service...international; varies widely by country and/or distributor.)  You should plan to receive your Packaged on...is launched, you’ll be able to work with the distributor to place a request and order if approved.  I ...

...on chemical (CaCl2, cationic liposome) or electrical (electric current forming membrane pores) transfection...virus. Virus purification can be a tricky process. You have to centrifuge the culture supernatant in order...The ability to infect non-dividing cells is not restricted to in vitro cell culture as lentivirus-derived...transducing non-adherent cells you might need to centrifuge your cells with the virus in order to aid viral...

...Restriction enzyme cloning is the workhorse of molecular cloning; however, one of its biggest limitations...that sequence modifications can only be made at restriction enzyme cut sites. The lambda red system is an... of DNA within E. coli and is independent of restriction sites. The lambda red system is derived from ...means cloning occurs in vivo, as compared to restriction enzyme cloning where the genetic changes occur...sections, key steps that differ from traditional restriction enzyme cloning will be explained in greater detail...screen for positive clones in most cases, and restriction enzyme digest can be used to screen plasmids ...

...This post was contributed by Kurt Swanson a structural biologist and protein engineer currently working...R206H point mutation on the receptor ACVR1. ACVR1 triggers appropriate bone regeneration in healthy patients...mouse models. As this biologic moves into clinical trials, there is new HOPE for AJ. But, as we in the scientific...fight against FOP in two ways. First, Addgene is distributing ACVR1 Plasmids from a variety of research labs...will be hosting the AJ’s Boston League team (Contributing many new heroes to the team in the process) ...

... a team to get a lot of stuff done.  Factors contributing to job satisfaction Being a good manager is ...mostly happy at work, they will work harder and contribute more. Assuming basic needs are being met (i.e...pay and benefits to live on), the factors that contribute to job satisfaction are a bit surprising. There...overwhelmingly agree that these are the factors that most contribute to job satisfaction: A feeling of being appreciated...organization and feel the importance of their contributions. Flexible work schedule Flexibility to manage...

...shut-down and the subsequent fluctuations in distribution. I don’t have space in this blog to call ...scientific community.  With the dramatic reduction in distribution, we had to figure out a way to maintain our ...operations until our normal work resumed. Addgene distributes and fulfills requests for scientists doing almost...especially proud to support Addgene now, when their distribution of plasmids for COVID-19 research is accelerating... cloning grade DNA service, and viral vector distribution is recovering from the pandemic-related slowdown...

...Human whole genome sgRNA iBAR library Article contributed by Susanna Bachle Figure 1: Initial steps...labeling using recombinase competition Article contributed by Aliyah Weinstein Sparse Predictive Activity...reporter for calcineurin activity in yeast Article contributed by Leah Schwiesow Calcineurin is a protein phosphatase...protein fusions as a DNA staining dye Article contributed by Jennifer Tsang Figure 4: Double stranded...single molecule-fluorescence assay to monitor restriction enzyme digestion of single molecules in real-...

... This post was contributed by guest blogger James D. Fessenden, an Assistant Professor at Brigham and... site when used in conjunction with Cy dye-nitrilotriacetic acid (NTA) conjugates (Kapanidis, Ebright,...50-69. Royal Society of Chemistry. 3. Ermolenko, Dmitri N., et al. "Observation of intersubunit movement...-mediated fluorescent labeling with (Ni2+: nitrilotriacetic acid) n-fluorochrome conjugates." Journal ...

...In addition to single plasmids, Addgene also distributes pooled plasmid libraries containing hundreds,...versatile offerings! We recently re-amplified our distribution stock of the Brunello Human gRNA library, and...processes we use to ensure high-quality library distribution. You can also use these tips as a starting point... scaled to 1, to create a “cumulative wealth distribution” curve, or Lorenz curve. By calculating the ...gain a sense of the uniformity of the library distribution, with an AUC of 0.5 representing perfect uniformity...

...immune response from the body. Several factors contribute to the immunogenicity of a drug, including the... body recognizes a drug as a foreign antigen, triggering a humoral immune response. ADAs can inactivate...one of the more common open-access tools, while EpiMatrix is a commercially available algorithm to evaluate...in silico assessments, as well as analyze the contributing factors that in silico prediction algorithms...monocytes. PBMCs can be further differentiated into dentritic cells or macrophages through cytokine induction...

...This post was contributed by Mary Tamer from BioBuilder, an innovative nonprofit bringing the study of... new for themselves and when they effectively contribute as part of a team.” As a seasoned educator, Kuldell...almost every state in the U.S. and in more than 30 countries around the world. Snapshots of the BioBuilder ...open-access curriculum begins with students reading comic strips and watching animations that present a specific... lab materials and reagents such as droppers, centrifuge tubes, circuit parts, and lab-strains of bacteria...

...Light-mediated block of synaptic transmission Article contributed by Klaus Wanisch  Listen to the optogenetics ...imaging of microbes in mammalian hosts Article contributed by Lukas Morgan  Listen to the acoustic reporter...and create ultrasound contrast. At a very low volumetric density and with a resolution of less than 100...mechanisms of miRNA-mediated gene regulation Article contributed by Michael Lemieux  Listen to the miRNA reporter...vivo using the CRISPR-Cas9 system CHIME Article contributed by Aliyah Weinstein  Listen to the CRISPR podcast...

...presentation, starvation or the use of caloric restriction mimetics trigger autophagy and, in a mouse model led ...deposit and distribution service! References: 1. Pietrocola, Federico, et al. "Caloric restriction mimetics...from a state of chronic hepatitis caused by hypernutrition and alcohol consumption. Chronic inflammation...

...collaborative group, she also joined early on and contributed her expertise in developing reproducibility workshops...Joanne Kamens, Addgene also had the opportunity to contribute to this open science collaboration. The first...workshop at the conference, but because each contributor made the materials accessible, anyone could present...present the material. For example, Addgene’s contributions by Tyler Ford included reagent sharing and were... upcoming conference or you’d like to join or contribute to this collaboration, please check out the official...

...worry about and that is something to recognize! Distributing plasmids, viral service, and more For the ...about succeeding at our core mission. Plasmid distribution is amazingly still growing at >9% every year...since 2005. We’re meeting the challenges of DNA distribution to ensure scientists everywhere can access plasmids... it. Almost 10,000 viral vector samples were distributed in 2018 and we are poised for more offerings....Addgene. There’s so much potential for service and contribution! Leadership On an organizational level, ...