...affinity purification with an anti-Cas9 antibody. These different tag systems and affinity purification schemes...genomic alterations. These studies utilize a catalytically inactive form of Cas9 known as dCas9 (Jinek ...interest targeted by a gRNA. CRISPR-mediated purification of a specific genomic region In 2013, the Hodaka...Subsequent mass spectroscopy studies allowed for the identification of proteins associated with the IRF-1 locus...steps (Fujita et al., 2013). CRISPR-mediated purification of a specific genomic region alleviates these...the initial report describing CRISPR-mediated purification of a specific genomic region, numerous adaptations...different fields of study. In addition to the identification of DNA binding proteins, this method can characterize...

...Technical Tips). Minigenome replication and transcription In order to replicate the minigenome, the viral...transcription and replication strategies of Marburg virus and Ebola virus by using artificial replication systems...allowing researchers to study aspects of the EBOV replication cycle under BSL-2 conditions (4). What is a ...that are crucial cis-acting signals for genome replication and viral gene transcription (6). The EBOV genome...5’ ends of the genome which are required for replication and transcription (Figure 1). All viral genes...cis-acting signals necessary to direct minigenome replication and reporter gene transcription by the EBOV polymerase... in 2, 5). The minigenome is capable of being replicated and transcribed in the cell when the appropriate...

.... Tracking samples - it's all about location, location, location There are many different types of samples...but we also have a dedicated and detail-oriented lab team that works carefully to catch mistakes. Each sample...assigning a physical location to your samples, include as much detail about that location as possible. Are...sample a unique location in a specific freezer will help ensure that samples are easy to locate in freezers...also help us keep track of the tubes’ physical location in the lab. These barcodes additionally allow ...Addgene. Each sample at Addgene has four layers of location which can all be extrapolated from the 2D barcode...Plate Freezer Rack Freezer Tubes are assigned a location within a plate that can hold up to 96 tubes (see...

...Series 4. Become a science communicator More and more fellowship applications are asking for a 200-300 ...good quality application. Even following this advice, only about 50% of my grant applications have been ...do need to be able to research and write your application, then give it to someone else to read and / or...it). It’s easy for reviewers to spot when an application is rushed (whether the idea isn’t properly researched...researched / developed or the application is full of typos) and these are unlikely to get past the initial...someone else in the department) to read over your application and give you feedback. They’ve most likely been...alterations before submitting - handing them your application 3 days before the deadline isn’t going to be ...

...in PubMed and choose the appropriate publication. This publication will be listed at the top of the final...classified in multiple categories, please check all of the appropriate categories (e.g., for a lentiviral... "Published plasmids" or "Unpublished or pre-publication plasmids" and set a desired distribution status...quickly associate your plasmids with the original publication where you described them. Search for your article...plasmids. You can submit "Unpublished or pre-publication plasmids": Unpublished plasmids or plasmids that...accepted. Addgene also encourages submitting pre-publication plasmids so they can be available when the paper...preprint submission, we are happy to update the publication information once the manuscript is published ...

...cleaved after purification, so as not to perturb protein function for downstream applications. Tag cleavage...thoughtful planning, affinity tags can make protein purification a cinch. Types of affinity tags, the proteins... Affinity tags are used primarily as protein purification tools (and can have secondary functions as detection...single protein is expressed, with the intent of purification, it will be expressed along with all other cellular...available antibodies, resins, beads, and other purification aids readily available. Choosing the best tag...tag to remain on your protein for downstream applications Below, we describe a few of the most common...cleavage must be performed after primary purification and requires a second clean up step to remove the cleaved...

...serially transfected using a cationic vehicle. The following synthetic modifications are used to protect the... to verify there’s no genomic integration Replicating EBNA1 episome Non-integrating Medium Medium ...creates heterogeneous iPSC cell lines, which can complicate comparisons made between lines. Incomplete silencing...Retroviruses are not considered safe for clinical applications.     Lentiviral: Lentiviral vectors infect dividing...lentiviral vectors insert in different genetic locations in each transduced cell, so there’s unavoidable...unavoidable heterogeneity between clones. This can complicate comparisons made between samples, i.e. transgene...extent of reprogramming could be affected by the location of insertion. Lentiviral insertion can also disrupt...

...ori2, ori3) and replication initiation proteins (RepA, RepB) necessary for replication in various hosts...the ori and allow for replication. However, if plasmids have their own replication machinery such as Rep...different hosts that a plasmid can transfer to, replicate in, and persist in. Some plasmids have a narrow... while others have a broad host range and can replicate and stably maintain the genes they carry among.... These features are: The plasmid origin of replication (ori) contains structural elements that are versatile...structure allows plasmids to utilize diverse host replication machinery. For instance, the ori typically contains... iterons that serve as recognition sites for replication initiation proteins. These iterons vary in their...

...their CV or grant applications. A number of organizations (such as ASAPbio) advocate for preprints, and...the most important part of your research is communicating the results of the work. Yes, those control ...any impact! There are lots of ways scientists communicate with each other: from formal mechanisms like ...poster or talk, or submitting patent or grant applications; to informal settings like lab meetings, Twitter...of the most significant shifts in scientific communication is the rising use of preprints, or scientific...publishing in a journal.  Accelerating Scientific Communication Preprints have been common in fields like math...of under a CC license. Many scientists and advocates have argued that while peer review is valuable...

...desired modification. Finally, the positive clones are sequenced to confirm the desired modification and ...polymerase activity (for amplification), 3’->5’ exonuclease activity (increases amplification fidelity), and no... genome modifications. Occasionally, multimerization of the PCR primers can cause duplication of the primer...the desired modification have been identified (and confirmed to be free of primer duplications), final validation...and the absense of additional modifications.   Site-directed mutagenesis of Plasmids. A) Generation..., which flank the targeted region. Upon PCR amplification, the template is removed by DpnI restriction...extension time to match the size of your template. Amplification of GC-rich plasmids is facilitated by the addition...

...methods of replication. In fact, an entire classification scheme, known as the Baltimore replication classes...that in a bit! Viral genomes and replication When it comes to replication, viruses tend to march to the ...developed around how they replicate (Figure 2). Figure 2: Baltimore replication classes. Image credit: ...an intermediary DNA replication step between DNA and mRNA. DNA genome replication can involve an RNA or...cell. The cell then uses its own machinery to replicate the viral genome and proteins. This is, for the...before being translated into proteins. RNA viruses replicate their genomes using a variety of RNA or DNA intermediaries... bacteria and eukaryotes, which use a binary replication process whereby one cell copies itself and “splits...

...begun my journey towards a career in science communication, an established field of inquiry with which ...University of Otago’s Department of Science Communication the following year and, having thoroughly enjoyed...straight on to a master's degree in science communication. The first year of the course involved a whirlwind...world of industry. Along this road, science communication graduates are often well-equipped. Many institutions...truly benefit others. Herein, a good science communicator—with an understanding of how to appeal to a ...pursuit of public engagement. Studying science communication is an equally fruitful endeavor for those embarking...towards academia. Many of those entering science communication (myself included) come from a scientific background...

...would be a good time to talk about the amplification and verification processes we use to ensure high-quality...your own experiments. Library amplification 101 Library amplification seems straightforward: E. coli ...transformed with the library DNA, they grow and replicate, and then their DNA is harvested, much like one...library has its own depositor transformation and amplification protocol, but the overall workflow is similar...ll need to remember when undertaking library amplification and sequencing. Maintain representation of the...electrocompetent Stbl4 cells when performing amplifications, and we use electroporation to ensure a good...your sequencing results represent a successful amplification? To provide a point of reference, Addgene commonly...

...potential applications of CRISPR/Cas9 in genome-engineering. One particularly attractive application involves... the perfect voltage indicator and optogenetic actuator pair. Ideally, indicators should possess fast ...random genomic location than to express the tagged protein from a defined genomic location or from its endogenous...temporal control over CRISPR/Cas9 mediated genome modification and gene expression. The plasmids associated... system widely accessible for many potential applications. Polstein LR & Gersbach CA, Nat Chem Biol ...other ions In BRET, A mutant Renilla luciferase, catalyzes the oxidation of its substrate, luciferin, which...amenable to both high and low throughput screening applications. The system can be readily modified to use different...

...them. An easy way to do this is gel purification. In gel purification, you use a voltage difference across...backbone. Sample results indicative of successful and unsuccessful ligations are indicated below. A successful...grow overnight cultures for DNA purification. The simplest purification you can do is a miniprep, but if...digests. Of course there’s much more detail and verification required for the process to work well, so let...backbone. Because you lose some DNA during the gel purification step, it is important to digest plenty of starting...prior to the ligation step or prior to the gel purification step, depending on the phosphatase you choose...   2. Isolate Your Insert and Vector by Gel Purification     Now that you’ve cut your insert and vector...

...you.  Starting from plasmids requires amplification, verification, and lentiviral production steps before...the same proportion after amplification as they were prior to amplification. And as anyone who has worked...process for using pooled lentiviral libraries. A) Amplification, validation, and lentiviral generation of a ...pooled library is transformed into E. coli for amplification. Transformed E. coli are allowed to grow on ...into HEK cells allows production of lentivirus. Purification of this lentivirus results in the pooled library...//pubmed.ncbi.nlm.nih.gov/27366891/   Amplification When you request a DNA pooled library from Addgene...might receive a similarly-sized aliquot from an amplification done by Addgene.) This small aliquot is not ...

...activity of CcdB. Applications of ccdB in plasmid cloning Perhaps the most widely used application of CcdB is...selection markers. Restriction modification systems Because restriction modification systems consist of two enzymes...used as biological tools for a wide range of applications in molecular biology or in vivo studies. Let...types of plasmid addiction systems and their applications. The discovery of toxin-antitoxin systems Toxin-antitoxin...CcdA (antitoxin) where CcdB inferes with DNA replication by binding the GyrA subunit of DNA gyrase and...construct. Other toxin-antitoxin systems and their applications MazE/MazF Aside from CcdB/CcdA, there are many...counterselectable marker to create unmarked genetic modifications in the yeast Pichia pastoris. By combining both...

...two types of replicate controls: technical and biological. In general, technical replicates can be thought... employ transfection, negative, positive, and replicate controls.  Transfection controls: empty vector... Plasmid B (as internal control) This result indicates that the transfection was not successful due to...the interrogation of the independent variable. Replicate controls: technical and biological Reliable results...derived from one source. Conversely, biological replicates ARE independent and can be thought of as "reproducibility...reproducibility controls". Biological replicates are what makes up your sample size (aka your n value)"... provide more in depth information. Technical replicates An example of a technical control is the transfection...

... 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 ...