... It generally stays put, which makes it easy to locate in a genome. But there is a type of mobile DNA,... hyperactive and likes to jump around from one location in the genome to another. This jumping is what...most transposable elements (TEs) results in the duplication of this target site sequence at the site of insertion...This is similar to how retroviruses, like HIV, replicate. Class I TEs do not encode a transposase enzyme...enzyme.  Class I TEs are considered replicative since every time they jump they make a copy of themselves...retroviruses both in their structure and mechanism of replication. They contain two genes: gag and pol. The pol...they move. Most Class II transposons have a non-replicative “cut and paste” mechanism of transposition: they...

...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 ...

...editors catalyze purine-to-purine or pyrimidine-to-pyrimidine base changes. Solid red arrows indicate a conversion...conversion directly catalyzed by the base editor. Dotted red arrows indicate the change introduced to ... point modifications. CRISPR base editing persists through multiple cell divisions, indicating that this...target modification in primary T cells, making them a promising tool for cell therapy applications. Dual...Base editing publication highlights In the table below, you'll find a list of publications we've highlighted...Early CRISPR applications were often limited by the low editing efficiency of homology-directed repair...following base editing and DNA repair. Black arrows indicate natural cellular processes. BER = base excision...

...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...

... your own research! Classification Structure Principles of CRISPR classification While CRISPR systems...Shakespeare for the first time. The discovery and application of CRISPR-Cas9 kicked off over a decade of frenzied...properties. This led to a need to develop a classification system for the array of CRISPR technologies...technologies. Learning the general principles of this classification system will help you gain a deeper appreciation...now seem neatly organized into their respective categories, arriving at a consensus was not as straightforward...multi-pronged analysis to sort effectively. The current classification system relies on a combination of sequence ...a similar function. After analysis, CRISPR classifications were placed into a fairly simple hierarchy:...

.... It sounds complicated but has a pretty low learning curve! Restraint: scruffing Location: throat Gauge... Restraint: mechanical restraint or anesthesia Location: tail vein or retro-orbitally Gauge: 27–30 Volume...practice to ensure the proper injection site has been located and to avoid potential off-target pokes to nearby...nearby organs. Restraint: scruffing Location: peritoneal cavity Gauge: 25–27 Volume: < 2–3 ml Skill required...getting a vaccine. Restraint: mechanical restraint Location: thigh Gauge: 25–27 Volume: < 0.02–0.05 ml Skill...Restraint: back scruffing or mechanical restraint Location: back or hind legs Gauge: 25–27 Volume: < 2–3 ...area must be shaved first. Restraint: anesthesia Location: between skin layers Gauge: 26 Volume: < 0.05 ...

...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...

...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...

...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...

...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...