... AAV.  Where do you start? Plasmids of course! You just need three plasmids to start making the AAVs you...packaging plasmid which contains the AAV structural and packaging genes, the adenoviral helper plasmid which...packaging plasmid and take a look at each of its parts. Key elements of the AAV packaging plasmid The packaging...packaging plasmid aka the RepCap plasmid encodes two genes: the replication (rep) gene and the capsid (... AAV RepCap plasmid! Check out this blog post to learn more about the AAV transfer plasmid! References... for the virus to replicate,  and the transfer plasmid which contains the viral genome. In today’s blog...lab, rep and cap are expressed from a separate plasmid in trans, freeing up space in the viral genome ...

...γ-retroviral packaging plasmids are not interchangeable. General envelope plasmids, such as VSV-G (Vesicular...in trans by specific plasmids. This method, therefore, reduces the number of plasmids that are required ...encoded by the transfer plasmid, but instead are provided in trans by other plasmids or the packaging cell...do many γ-retroviral plasmids have similar names? The nomenclature of the plasmid backbone is meant to...components are needed: γ-Retroviral transfer plasmid encoding a transgene, sgRNA, or shRNA of interest..., which facilitate integration of the transfer plasmid sequences into the host genome. Typically, it is... genome upon viral transduction. Most transfer plasmids available from Addgene were derived from MoMLV...

...move antibiotic resistance genes between plasmids or from plasmids to the bacteria’s genome. In humans, if...activity (Voigt et al., 2016). Sleeping Beauty plasmids  piggyBac Although its name suggests otherwise...codon-optimized wild-type piggyBac transposase.  piggyBac plasmids    Tol2  Tol2 was the first reported active DNA...increase in transposition activity  Tol2 transposon plasmids    Applications of transposons Now that you’ve... 9:135. https://doi.org/10.12688/f1000research.21018.1 Munoz-Lopez M, Garcia-Perez J (2010) DNA Transposons...

...blog Plasmids 101: protein expression Plasmids 101: E. coli strains for protein expression Plasmids 101...mutation” field on the plasmid page (see plasmid 87904 for example). As many plasmids available from Addgene...codon optimized by ordering plasmids containing them from Addgene. If a plasmid at Addgene contains a gene...

...Addgene blog Plasmid 101: Inducible Promoters Plasmid 101: Repressible Promoters CRISPR 101: Which Cas9... a gene (cDNA) can be done by either plasmid or virus. A plasmid carrying your cDNA can be targeted for...knock out. Looking for some permanent knock out plasmids? Check out our Cre vector and Murine LoxP vector...of protein expression. Addgene has a dCas9 KRAB plasmid for transient knockouts!  Turn it on Turn it on...

... How to Use the Nanobody Toolbox Find Nanobody Plasmids Structural comparison As the name suggests, ... Species Antibody Subclass Nanobody Addgene Plasmid Rabbit N/A* TP897 pTP1183 (1x cysteine) pTP955...Nanobody In Bacteria: Table 2 also has a link to the plasmid you’ll need to express your nanobody of choice ...column. bdNEDP1 protease can be generated using plasmid pDG02583. Label the Nanobodies: Purified nanobodies...Journal of Cell Biology. PubMed PMID: 29263082 Find plasmids from this publication at Addgene. 2. Bates, M... on Addgene.org Find more nanobody expression plasmids here Additional Resources Looking for monoclonal...

...the plasmid based on the predicted sizes and organization of different features within the plasmid. Restriction...if you don't have the full plasmid sequence. Once you have purified plasmid DNA, this method can be done...example plasmid on the right has a total size of 7.3kb, including a 1.2 kb insert. The plasmid was digested...possible plasmid conformations, with relaxed and nicked marked with asterisks (*). When the plasmid is digested... Congratulations, you have a plasmid expressing your gene of interest (YGOI) and are ready to dive into...functional experiments! Whether you’ve cloned the plasmid yourself or obtained it from a colleague down the...with the correct construct, and verify that the plasmid you received matches the expected sequence. Here...

... Addgene Blog How to verify your plasmid Check out our Plasmids 101 featured topic page Additioinal ... we use SnapGene for plasmid quality control are: To identify important plasmid features To align NGS...design experiments for further plasmid verification Identifying plasmid features While our quality control...control process for incoming plasmids can vary based on the contents of a plasmid, the first step is always...send the plasmid off for Sanger sequencing.     If a repetitive region is present in the plasmid, we might...sequencing results for plasmids in the repository at a glance. Our Snapgene-powered plasmid maps and sequences...results to perform quality control on deposited plasmids. We’ve also talked about how our new Snapgene ...

... Read all of our Plasmids 101 blog posts Check out all of our blog posts on plasmid cloning Resources...don't exactly "eat" plasmids as depicted in this comic, when a bacterium uptakes a plasmid, the genes on the... the plasmid can give them different phenotypes. For example, this plasmid encodes a green fluorescent...comes from adjacent lysed bacteria and can include plasmid DNA or fragmented DNA released into the environment...membrane so the bacteria can easily uptake your plasmid of interest. Scientists can also use electroporation...exist as transient extrachromosomal DNA, like a plasmid, or it can integrate into the host bacterium’s ...cloning vector that contains both bacteriophage and plasmid properties. The phagemids are packaged into replication-incompetent...

...single plasmid transformation, though, a pooled library has hundreds to millions of distinct plasmids, all...anyone who has worked with individual plasmids knows, some plasmids amplify well and others don’t. The size...pooled library, which contains a collection of plasmids in a single tube, or a pre-made viral prep library... prepare the libraries for you.  Starting from plasmids requires amplification, verification, and lentiviral...library and appropriate packaging and envelope plasmids into HEK cells allows production of lentivirus...amplify the library. This is similar to amplifying a plasmid: transform E. coli with the pooled library DNA,...own protocol that amplifies without disrupting plasmid representation. A good place to start is with a...

...hold the plasmid together, even if there are gaps or flaps remaining. This incomplete plasmid looks like...cloning uses short sticky ends to insert genes into plasmid backbones, base pairing between these ~4 base overhangs... overhangs isn’t strong enough to hold a plasmid together through transformation, so the cloned vector...longer single-stranded overhangs can hold a nicked plasmid together long enough to get it into cells and let... of LIC is often limited to specially designed plasmids. Sequence- and ligation-independent cloning: A...fixing up the “damage” and producing a complete plasmid. Besides being compatible with any vector, the ...into bacteria, which will efficiently repair the plasmid. Created with BioRender.com.   SLIC also opened...

...Additional Resources on the Addgene Blog Browse All Plasmids 101 Posts Learn about Mammalian Vectors Read about...Once you've gotten to know what a plasmid is in general, it's time to take a look at some of its parts...origin of replication are necessary to replicate plasmids and make sure the cells maintains it. Here, we'll...the promoter, which is necessary for getting the plasmid express our gene of interest. This element is responsible.... This means that the promoter present in your plasmid backbone must to be compatible with the type of...transcript, you will also need to make sure your plasmid has a promoter suited to working in your host organism...development-specific promoters available to scientists. Plasmids are oftentimes put to therapeutic uses, and in...

...Production at Addgene Viral Vectors 101: What is a Virus? Viral Vectors 101: Viral Vector Elements ...typically transfected along with viral packaging plasmids into a viral production cell line, usually HEK...HEK-293 cells. When all of these viral plasmids are expressed within the production cell line, viral particles...

...of a homologous donor (e.g., sister chromatid, plasmid, etc.), and consists of three pathways: classical...disrupted in the donor. Choosing a type of donor: plasmid, oligo, double or single stranded? The size of ...longer than 200 bases can be difficult, thus dsDNA plasmids are generally preferred for large insertions such...between 500-1000 bps. HDR efficiency with circular plasmid templates is generally low; to increase the frequency...edits, researchers have designed self-cleaving plasmids that liberate the targeting region from the vector...these are generally more toxic to cells than their plasmid counterparts. Another consideration for dsDNA templates...-depth information on HDR and CRISPR. Our CRISPR 101 series was designed to help explain the basic principles...

...Viral Vectors 101: Viral Vector Elements Viral Vectors 101: Pseudotyping Viral Vectors 101: Systemic Capsids... ITRs are the elements that make an AAV plasmid an AAV plasmid.  Within the AAV genome, ITRs serve as...gene of interest in a cell or animal model. AAV plasmids contain several important elements, but among ...molecule as the gene that they package. In a transfer plasmid, the transgene is flanked by two ITRs, and the ...genome of AAV in the native virus. In an AAV vector plasmid, they flank the gene of interest. A single ITR ...growth culture and result in a high percentage of plasmid DNA with mutated ITR sequences. Using recombinant-deficient...located immediately outside of the ITRs in the plasmid backbone and do not properly assemble, but these...

... consists of a Ti plasmid, into which the GOI is inserted, and a Ti "helper" plasmid, which contains Vir...for creating plant expression plasmids, including plant CRISPR plasmids from the Yiping Qi lab and the...plant-specific modifications to commonly used CRISPR plasmids are necessary to use the CRISPR system in plant...et al., 2013). Addgene carries Cas9-containing plasmids for knockout, activation and repression of target...expression of both Cas9 and the gRNA off of the same plasmid. Delivering CRISPR components to plant cells Once...and insertion into the plant genome. B) The Ti plasmid and helper are transformed into agrobacterium tumefaciens...and exposed to plant cells. The region of the Ti plasmid between the T-DNA borders (yellow) are recognized...

... can clone the genes from the hybridoma into a plasmid to create a recombinant antibody.     Figure...Resource on Addgene.org Check out Addgene’s antibody plasmid collection Read the latest on Addgene's new antibody...

... Antibodies 101: Immunoprecipitation  Antibodies 101: Polyclonal Antibodies Antibodies 101: Introduction...Antibodies Resources on Addgene.org Find plasmids encoding recombinant antibodies at Addgene ...

...packaging plasmids: the pHelper plasmid containing Ad E2A, E4, and VA RNA genes; and the pRepCap plasmid containing...with your transfer plasmid, carrying your gene of interest, and the two helper plasmids, commonly called...called “triple plasmid transfection” (Figure 1).    Figure 1: Overview of key steps of AAV production, iodixanol...repeats (ITR). You should always have a high-quality plasmid standard and rAAV reference material of a known...before undergoing titering, to remove residual plasmid DNA carried over from the production process. For...common AAV applications by reading our Viral Vectors 101 series!  This post was originally written by Didem...blog Parts of the AAV Packaging PlasmidParts of the AAV Transfer PlasmidAAV Variables That Matter AAV protocols...