...block of our understanding of complex biological processes. Many tools exist to perturb biological function... interference (RNAi) is a conserved eukaryotic process where approximately 20-30 nucleotides of double-stranded...formation of a hairpin RNA structure, resulting in processing by the endogenous RNAi machinery. Alternatively... engineered to created artificial miRNAs. The processed miRNA or siRNAs then silence the target gene(s... In plants, different dicer-like enzymes (DCL) process the dsRNA into specific types of interfering RNAs...cleaved mRNA can induce further rounds of RNAi. This process, known as “transitivity,” calls into question any...variable silencing efficiency and labor-intensive processes to isolate RNAi plants. With a visual reporter...

...orientation of the FRET pair. Since many biological processes occur within the typical FRET range, FRET efficiency...fluorophore on the same protein and are useful when a process affects the conformation of the probe. More on ...answer sophisticated questions about cellular processes. Currently, FRET experiments can probe protein-protein... activity of small molecules, detect cellular processes and signaling cascades, quantify mechanical tension... 2015), which may lead to monitoring of these processes in live animals (Hirata and Kiyokawa 2016), and...correlated information available from a cellular process. FRET experiments are expected to continue to contribute...insertion within green fluorescent proteins." Proceedings of the National Academy of Sciences 96.20 (1999...

...over from the production process. For more details on the qPCR titering process, refer to the Addgene AAV...contamination, use appropriate decontamination procedures (Korte et al., 2021) for all disposable items...practices diligently throughout the production process.  Helper-free production Unlike lentivirus, AAV...supernatant and/or producer cells for further processing. What component(s) you need to collect will depend... if needed. For more details on the production process, check out the Addgene AAV Production protocol....Protocol. If you’re interested in the ddPCR titering process, which is what Addgene uses, check out our ddPCR...

...inhibitor of base excision repair (BER), the cellular process that catches mutated U’s in DNA and ideally converts... In this case, the team suspected that the BER process was excising the incorrect U base, creating an ...editor. Black arrows indicate natural cellular processes. Created with BioRender.com. Beyond UNG: Optimizing...The researchers hypothesized that the CGBE edit process occurs entirely while Cas9 is bound, keeping the...intermediate of CGBE activity, and they are sometimes processed by error-prone non-homologous end joining (NHEJ...DNA abasic sites, protecting them from cellular processes that would cause DSBs, and its inclusion significantly...Non-Homologous End Joining An error-prone cellular process to repair DSBs. Base Editing terms   Base transition...

...trickier -- but at Addgene, we try to make the process as smooth and simple as possible. We are the Customer...frequently asked questions related to the ordering process: I placed an order through my university’s purchasing... directly from your website? Many vendors will process orders based on a purchase order, which is an official...the legal agreement (MTA), which streamlines the process and saves you time. Anyone affiliated with the ...you have additional questions about our ordering process not addressed in this article, check out our Help...answers to common questions about the ordering process, the Addgene Help Center is full of information...

...After a bit of research and thinking about my own processes, I’ve broken down time management into three conceptual...conceptual chunks: prioritization, planning, and procrastination. Time management through prioritiziation Every...Eric before doing anything else. Preventing procrastination Even if you’ve done all the prioritizing and...and planning in the world, procrastination can easily prevent you from making progress on a task. For ...few things I’ve found useful to avoid such procrastination: Remove distractions: Probably the easiest...STARTED - One Harvard Business Review article on procrastination recommends testing out how much time you can... procrastination...

...that has defined our understanding of biological processes and developments. Model organisms often feature...history and characterization. Many proteins and processes naturally found in E. coli have been manipulated.... coli can be used as a model to relate these processes in more pathogenic bacteria (Blount, 2015). Rattus...McCusker and Gardiner, 2011). As many biological processes and signaling pathways that regulate regeneration...humans. Thus, understanding their life cycle and processes with their regenerative abilities is expected ...the most significant and fundamental biological processes. Many of their genetics and developmental patterns...Wang, S. H., & Jiang, J. P. (2016). Research proceedings on amphibian model organisms. Dong wu xue yan...

...to track or measure the process you’re interested in. Depending on the process you would like to monitor... sensors’) are biological tools that monitor a process or detect a given molecule. The sensor component...over time. You can also use biosensors to follow processes such as protein or DNA binding, enzyme or transcriptional...  Detection Method Notes Quantification of a process Plate-based assays (plate reader) Detect the average...of cells (i.e. in a plate well). Use to track a process (e.g. enzyme activity) over time or under various...

...gels to nitrocellulose sheets: procedure and some applications. Proceedings of the National Academy of Sciences...  Figure 1: Overview of the Western blot process. The sample, a mixture of protein is separated ...antibodies, and finally visualized.   This process allows you to detect a single, specific protein...conduct the electric current. The wet transfer process is very effective in transferring a broad range...common readout for subcellular fractionations, a procedure that isolates proteins from different cellular... antibody specificity and antigen structure. Proceedings of the National Academy of Sciences 76:3116–3120...

...replenishing kits, is to find new ways to make lab processes more efficient. Being in the Boston area, we get...workshops and can use what we learn to improve processes in the Addgene lab. In this post, I’ll tell you...sample into many small components. Without proper processing, these small DNA strands can form concatemers...libraries from previously fragmented DNA.The general process for library preparation using this kit is: Fragmentation...that regardless of how much DNA we started the process with, we got great amplification of our sample ... for us as a company to constantly update our processes and get new ideas from learning experiences like...

...content. Regardless of the process, everything goes through an editing process and final check before publication...invite you behind the scenes in our content writing process, and to even get involved yourself. You probably...science, and our entire mission is to enable that process. Our repository of plasmids, AAVs, antibodies, ...recommendations. A few discussions in the review process led to quick dives back into the literature to ...and hit publish. Making content is an iterative process. We are constantly reviewing the resources we currently...

...biological sources of DSBs, and which need to be processed. Processing occurs prior to ligation as incompatible.... After the initial cut, the next steps in the process involve repairing chromosomal DSBs. It is important...step. Accordingly, NHEJ has a vast toolbox of processing factors, including polymerases (Pol μ and Pol...Although we do not describe these steps here, the processing of DNA ends tends to be the point where mutations...Non-Homologous End-Joining Really an Inherently Error-Prone Process? PLoS Genet 10:e1004086 . https://doi.org/10.1371...

...guide to our online data entry process. Figure 1: Begin the deposit process by clicking “Submit Online Deposit... our deposit spreadsheet?  Our plasmid deposit process can be broken down into four simple steps: Send... contact you if any issues arise during the QC process. As the depositing scientist, you will have the...information in a deposit spreadsheet. The data entry process On the Addgene home page, if you scroll over “Deposit... plasmid deposit process...

...cloning processes will require a PCR reaction or two to generate templates for the cloning process. Time... products via overhangs left over from the PCR process (the precise mechanism isn’t well understood). ...backbone. Once combined, the cut and ligation processes happen in a matter of minutes. Cost: Funding ...commercially available cloning kits can often make the process much more streamlined, because they will come with...reaction in favor of the desired assembly to make the process close to 100% efficient. Gateway cloning also offers...

...quality, we undertake a rigorous quality control process. In 2016, we launched our viral service to continue...bacteria snuck into the prep during the production process? These questions are especially important when ...potential contaminants. The analysis is a 2-step process using Geneious software: First, the individual ...CHRM4 gene. When contamination is suspected, we proceed to de novo assembly of the unmapped reads, employing...validate our method. We then extracted the DNA and processed the mixed sample in our pipeline (see Figure 2...answer your questions about our quality control process! The images in this blog post were created using...

... be tracked easily at each step in the storage process. After the original sample is barcoded, the lab...stocks, prepped DNA ready for our quality control processes - all of these samples have the 2D barcodes mentioned...a rack of tubes is dropped at any point in the process, we know exactly which location each should go ...step in the receiving, transforming, and storage process, we ensure that the sample tubes that we use match...Addgene’s secret to success. It helps us quickly process samples accurately and in a way that can be easily...deposited samples along every step in the storage process - from deposited DNA to transformed cells and finally...

...the grant development and management process or the whole process, including grant readiness, funding ... that likelihood. We provide expertise on the processes needed to successfully implement, manage and close-out... funds available and the increasing regulatory process for grants has fostered the demand for experts ...position that required some involvement in the grants process and managing programs that were grant funded. I...and experience through the candidate application process, the candidate’s ability to write a grant application...

...selection process to standardize our feedback and evaluation of viral vectors. This process builds on ...significant time and removes a variable from the lengthy process of validating and using an AAV in a specific experimental...its inherent bias: Addgene uses a prioritization process to decide which tools we make available. This ...us first identifying broadly useful vectors, a process that isn’t as simple as it seems. Take, for instance... and blog posts. The lessons learned from this process have shaped and supported our growth and development...

...ELISA basics All ELISAs follow the same overall process. First users coat a multiwell plate with a capture...cross-react with samples, eliminating them from the procedure removes one potential source of cross-reactivity... labs can conjugate antibodies themselves, the process can be laborious and expensive. In some cases, ...the bench.   Indirect ELISAs The indirect ELISA process is similar to that of a direct ELISA, but the indirect...antibodies that work well together. Moreover, the procedure is longer than alternative ELISA methods, especially...peptide. The positive control will confirm that the procedure is working and should always be included.  For...

...control point of DNA replication in bacteria, so the process is carefully regulated by a mix of positive and...replication (oriC), where initiation begins. This process relies on the initiation protein DnaA. First, DnaA... versions is required to start the replication process. When enough copies of ATP-DnaA are bound, including...integration host factor (IHF) assists with the process: it bends the DNA to redistribute the DnaA from...plasmid ori, after which replication of the plasmid proceeds (and terminates) similarly to the bacterial chromosome...complexes between iterons on different plasmids in a process called "handcuffing," which blocks replication....