...Student at the University of Manitoba. Location, Location, Location! Failure to Plan (for Storage) Is Planning...Viral Vector Blog Posts from Addgene 1. Location, location, location! What do buying real estate and using...for a house and choosing an AAV are… location, location, location! Just like this common real estate mantra...important factors related to location. Location (1): What tissue? Location (2): Where in the tissue/which...which cell type? Location (3): Traveling from the original location - Axonal Transport in the CNS What...particular experiment! Traveling from the Original Location - Axonal Transport It has been demonstrated that...previous literature using AAVs for DREADD delivery indicates ~7-21 days are required for the onset of gene ...

...Each round of amplification should result in a 10-100-fold increase in virus. Purification (2 days) - Purification... Purification is required if rAdV is to be used in vivo. The standard method for purification of rAdV ...vectors (AdV) are attractive vectors for research applications and gene therapy: they can be produced at high...AdVs is what makes them ideal candidates for applications in oncolysis and vaccination!) In the 65 years...Adenovirus-mediated gene delivery: Potential applications for gene and cell-based therapies in the new...genes E1 and E3. Without these genes, AdVs cannot replicate on their own but can be produced in E1-containing...The ever popular pAdEasy system belongs to this category. 3rd Gen AdV (Gutless or high capacity AdV) are...

...lab while the publication page contains all the plasmids deposited from the same publication. Next up, we...number given to every plasmid in our catalog - if you know the catalog number of the plasmid you are searching... Addgene’s plasmids is an indication of popularity. A yellow flame indicates that a plasmid has been ordered...through the depositing lab page, then onto the publication page, and finally on down to the individual plasmid...deposit process and these can be found in this location as well. These maps make it easy for scientists...Great! Now, if I want to use this plasmid in a publication, how do I know who to acknowledge? Depositing...plasmids that are available through Addgene. Publication: If the plasmid has been published, the paper...

... ITRs serve as the origin of replication and are required for replication, packaging, and vector persistence...viral rep (replication) and cap (capsid) proteins, which bind to the ITRs and initiate replication. ITRs are...structures, ITRs are prone to replication errors and disruptive mutations and truncations that can make vector ...binding site for Rep78 and Rep68 to initiate replication and a terminal resolution site that serves as... are noted. Created with biorender.com. ITR modifications and types  Since ITRs are one of the most important...important elements of AAV and rAAV vectors, modifications can drastically affect gene transfer efficiency... Rep proteins are important for viral genome replication, these mutations can affect transduction efficiency...

... be run in duplicate or triplicate. The closeness of the data points generated by replicate values provides... the standard dilution series in duplicate or triplicate. Replicates allows you to calculate the curve... deviation of each replicate measurement divided by the mean of all the replicates multiplied by 100. ...regularly, and mix samples very well.  Use duplicates or triplicates to increase the accuracy of your experiment... critical for a number of popular scientific applications, and the quality of a standard curve can make...diluted standard as a function of concentration on a scatter plot. For the BCA example, the concentration of...correlation between the actual values and the predicate values and measures how well the data points fit...

...of its biggest limitations is that sequence modifications can only be made at restriction enzyme cut sites...homologous recombination. It allows for direct modification of DNA within E. coli and is independent of ...engineering.  It can be used to make an assortment of modifications: insertion and deletion of selectable and non-selectable...expressing the lambda red enzymes. These enzymes then catalyze the homologous recombination of the substrate ...however, only Beta is required when generating a modification with an ssDNA substrate.  Gam: Gam prevents ...regions flanking the target site).  The best applications for dsDNA inserts include: large insertions ...nucleotides long with the desired alteration(s) located in the center of the sequence. Since lambda red...

...your specific application. Consider also that there may be variations within an application. Take immunohistochemistry...website highlights a variation of your application, a publication successfully used it in a similar way...choosing an appropriate antibody first look at the application-specific data provided on the manufacturer’s ... a linear antigen tend to work very well for applications that denature a protein to its primary (linear...the most similar to yours. Take a look at the publications cited on the manufacturer’s website, which may...plan to. To secondary or not to secondary?  The application will also determine whether you should use an...antibody. If you are trying to visualize the precise location of a protein in a tissue section then a directly-conjugated...

...recombinase catalyzes the site specific recombination event between two loxP sites, which can be located either...maintained).  Translocation: If the sites are on separate DNA molecules, a translocation event is generated...important plasmid elements – promoters, origins of replication, protein tags, and antibiotic resistance markers...creating (excuse the pun) specific, targeted DNA modifications in transgenic animals, embryonic stem cells,...proteins or co-factors, thus allowing for broad applications in a variety of experiments. LoxP (locus of ...and the canonical loxP sequence is ATAACTTCGTATA-GCATACAT-TATACGAAGTTAT. The loxP sequence does not occur...Therefore, inserting loxP sites at deliberate locations in a DNA sequence allows for very specific manipulations...

...which provides the AAV replication (rep) and capsid (cap) genes. AAV replication uses the host’s polymerase...this AAV purification video! It didn’t exist when I did my first iodixanol gradient purification, but I ...In this post, I’ll share some AAV production, purification, and titration tips, while also summarizing ...adenovirus genes which help mediate AAV viral replication; and 3) the transfer plasmid which encodes a ... that aids in the homologous recombination of duplicated regions like the ITRs. Restriction digest screening...consider performing PEG precipitation. Instead of sonicating the cell pellet, I lyse the cells with this AAV... with benzonase immediately before iodixanol purification. This approach gives me a bit of flexibility...

...(stay tuned for our affinity purification tag blog!).   Applications of epitope tags   The...success rates across all applications. You can find FLAG antibodies on Addgene’s catalog including a mouse monoclonal...protein purification is not ideal with Myc, as low pH conditions are required during the purification, which...experienced. Finding an antibody that works for your application, specifically detects your protein, is species...and have been widely used across species and applications. In this blog, we will review the most common...that are bound by antibodies for the purpose of purification or detection of the tagged protein. These peptides...not exist, or they don’t exist for a specific application, or they exist but are unreliable for a variety...

...not cut within your insert Are in the desired location in your recipient plasmid (usually in the Multiple...amplify and design primers that will bind to and replicate it. The following image shows the ends of the ...Forward Primer will use the sequence 5'-ATGTGGCATATCTCGAAGTAC-3' for the region that binds the ORF and...primer, making our Forward Primer 5'-GAATTCATGTGGCATATCTCGAAGTAC-3'. Many restriction enzymes do not cut...final Forward Primer sequence of 5'-TAAGCAGAATTCATGTGGCATATCTCGAAGTAC-3'. For the Reverse Primer, the design...need to use the reverse complement to get PCR amplification. We can start similarly, taking the final 18...of the ORF, including the stop codon (5'-TGGCATATCTCGAAGTACTGA-3'), then adding NotI (GCGGCCGC) and then...

...3’ end binds template DNA for PCR amplification, while modifications and homologous sequences are encoded...multiple modifications A key feature of IVA cloning is the ability to combine multiple modifications. Any ...’ Check your amplification by gel electrophoresis. In our experience, good amplification after PCR means...gain traction? It is likely that both vector amplification with low fidelity polymerases and the cost of...this approach requires no special reagents or purification kits, it is low cost and can be immediately ...to produce the linear fragments, as desired modifications and homologous regions can be encoded in the...are perfectly fine, yet performing multiple modifications will require ultracompetent (109 CFU/μg) cells...

...far, we’ve used the MUCICAT system in two ways: In our initial paper, we used MUCICAT to optimize the gene...industrial strain. Not only is MUCICAT better, it is faster. Using MUCICAT only needs 2 newly constructed...integration using CRISPR-associated transposases” or MUCICAT. Here, we’ll describe each of these CRISPR systems...providing users with a complete editing process. The MUCICAT system While the above systems are great for experiments...chromosomal integration (ex: genome editing), our MUCICAT system which is based on the CRISPR-associated ...unstable and lack precise control over copy number. MUCICAT system contains 4 plasmids: pDonor, pTnsABC, pQCascade... crRNA can be customized as well, including concatenations into crRNA arrays targeting multiple different...

...This application of AHA labeling is known as fluorescence noncanonical amino acid tagging (FUNCAT, Dieterich...experiments. Visualizing newly synthesized proteins with FUNCAT Professor Schuman’s technique applies metabolic...the whole de novo synthesized proteome. However, FUNCAT alone cannot be used to determine the turnover ...specific protein. Professor Schuman’s lab showed that FUNCAT’s usefulness can be extended to determine the turnover...ligation assay (PLA). The clever combination of FUNCAT and PLA PLA was originally published by the Söderberg...DNA strands take part in rolling circle DNA amplification. The amplified DNA segment is labeled with fluorescent...fluorescent probes that enable visualization of the location of the protein-protein interaction. To study protein...

...from other sources. Mode of communication Different modes of communication take up different amounts of...your audience needs to understand what you’re communicating. While I’m sure that there are loads of ways...new languages!) Jargon has a place in science communication, but the more you use, the higher cognitive ...This can be obvious, in that a lot of science communication is helping people understand new concepts. You...intuitive, or logical to a layperson. When you’re communicating ideas that you think are clearly connected, ...pick and choose what concepts and details to communicate. It’s okay (and encouraged) to leave blank spaces...over some aspects when needed. The trick is to communicate your key points well enough that the audience...

...Description Origin of Replication (ORI) DNA sequence which allows initiation of replication within a plasmid...point for PCR amplification or sequencing. Primers can be exploited for sequence verification of plasmids...important. Our “Plasmids 101” series designed to educate all levels of scientists and plasmid lovers - serves...plasmids are small circular pieces of DNA that replicate independently from the host's chromosomal DNA....virulence. All natural plasmids contain an origin of replication (which controls the host range and copy number...Minimally, lab-created plasmids have an origin of replication, selection marker, and cloning site. The ease... plasmids and the ability of plasmids to self-replicate within a cell make them attractive tools for the...

...of complex post-translational modifications. Extraction and purification of recombinant proteins from ...modified via an array of post-translational modifications including protein folding, formation of disulfide...mRNA and the addition of post-translational modifications Learn more molecular biology in our Plasmids... experimental, biotechnological, and medical applications. Researches can visualize proteins in vivo by...expression systems used for protein production and purification. These include mammalian, insect, bacterial,...separated from contaminants using a variety of purification methods. Deciding which expression system to...protein you need   Your plans for downstream applications In this blog post we will summarize some of...

...results.  Replicates Replicates are another critical control in biological experiments. Replicates can be...of biological replicates such as multiple mice in each group and C, technical replicates or repeated measurements...secondary antibodies.   Application-specific controls Some antibody-based applications have very specific ...then moving on to controls for antibody-related applications. After reading, you will have a better understanding...blot and look for the molecular weight shifts indicative of tau phosphorylation. By including the positive...statistical power of an experiment. Technical replicates are repeated measurements of the same sample ...difficult to obtain meaningful results.  Biological replicates are distinct samples that are treated identically...

...Science Communication Conversation in Science Communication The Purpose of Science Communication #1 Mistake...supporters of science communication. To help you think about ways to effectively communicate your science, we...News at Harvard University. Effective science communication is required to promote public support for research... organization that works to promote science communication. Science in the News hosts a podcast, seminars...That’s right, #ActualLivingScientists, eager to communicate their science. In this first half of our two-...programs SITN runs and learn how they think about communicating science. Listen to the interview with Science...Mistake Scientists Make when Communicating with Non-Scientists How to Approach Different Audiences Listen...

... information you are looking for in our catalog or educational resources: plasmid, pooled library, blog...articles. From paper to plasmid Authors regularly indicate that their plasmids are available from Addgene...papers. Maybe they started their deposit prior to publication and were able to include specific plasmid ID ...terms we can use to find materials from this publication. Searching for the title of a paper works You...filters Not every plasmid search will start from a publication. You may have an idea for a plasmid that would...try different terms you may find the available categories and filters change as we now only show options... a second filter, this one from the ‘Requests’ category, will leave us with a single plasmid to consider...