...consisted of three plasmids: the packaging, envelope, and transfer plasmids. The packaging plasmid carried a mutated... packaging and envelope plasmids are two of Addgene’s most requested plasmids! Lentiviral particles with...Many CRISPR plasmids are designed for lentiviral use, including Addgene’s top requested plasmid in 2015: ...itself due to a few missing proteins. The envelope plasmid contained a viral envelope to dictate the tropism...the vector could infect. Finally, the transfer plasmid encoded the desired transgene flanked by HIV-1 ...genome integration. After co-transfection of these plasmids, 293T cells released transgene-containing lentiviral...recombination between the transfer and packaging plasmids does exist. To further reduce this possibility...

... requested plasmids. I wondered how many scientists have used the protocol for this plasmid, and if it... your protocols onto your plasmid page, scientists who request your plasmid will have easy access to your...restriction digest of plasmid DNA, and bacterial transformation. And more plasmid-related educational resources...be found in our Plasmids 101 blog series. Want to learn more about sharing your plasmids via Addgene? Check... resources, such as protocols, Plasmids 101 articles on various plasmid features, and reference tables...deposit their plasmids to the repository. These protocols show up on the related plasmid pages and can... protocol is being used around the world? In the last month it’s been viewed by users in the US, China...

...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...

...recommended for plasmids up to 25 kb, most cloners should be fine, especially using plasmids in the 10-12...Other Plasmid Cloning Techniques like SLIC and Golden Gate Cloning Learn How to Verify Your Plasmid Read...Reference Pages Choose Your Perfect Plasmid Backbone Find Plasmids for Your Research ...made genomic SDM relatively straightforward, but plasmid-based SDM has lagged behind. While commercial kits... REPLACR (Recombineering of Ends of Linearized Plasmids After PCR). Current site directed mutagenesis...mutation, then use those to PCR amplify the template plasmid. The parental, nonmutated DNA is digested with ...of background colonies carrying the unmodified plasmid. For insertions, it’s possible to use the same ...

...introduce you to the plasmid class covered on that page. To help you find popular plasmids, we’ve also added...changes we’ve made: New CRISPR plasmid pages We’ve given our CRISPR Plasmids and Resources a fresh new design...design, sorting the plasmids we have available by category. Each plasmid page now has a helpful graphic... new plasmid pages that cover some of the hottest CRISPR tools. Base Edit: Base editing plasmids allow...revolution, Addgene has distributed over 100,000 CRISPR plasmids. But that’s not our only job - we strive to also...up with new developments (and, of course, find plasmids that will be useful to you.) With these goals...mind, we set out to update our web resources and plasmid pages to make them more useful and easy to navigate...

... and A.B. Lassar, Expression of a single transfected cDNA converts fibroblasts to myoblasts. Cell, 1987... [6], plasmid electroporation (episomal) [7, 8] and mRNA transfection [9]. Many of the plasmids used for...cells [18] Red blood cells [19, 20] Osteoclasts and osteoblasts [21, 22] Cardiomyocytes [23, 24] Smooth...treatment of mouse fibroblasts with 5-azacytidine led to their conversion into myoblasts [39]. In 2000, Ferber...Jeon, O.H., et al., Human iPSC-derived osteoblasts and osteoclasts together promote bone regeneration in...can be easily isolated from donors. Apart from fibroblasts, human keratinocytes from hair pluck, peripheral...them into different cell lineages [13]. Over the last decade, many cell types have been successfully differentiated...

...about Popular Plasmids in the Repository Learn about New Plasmid Technologies in our Hot Plasmid Articles ...to create a plasmid based on the biotin ligase BirA backbone (Eric Campeau, Addgene plasmid #26624) was...inserting BirA into a pMal plasmid (NEB) thus creating the pMal-T-Avi-His/BirA plasmid now available through...other Hot Plasmids Find Additional Posts on Mol Bio Techniques Find Additional Posts on Plasmid Technologies...controlled easier than formation of GST dimers. Avi-Tag plasmid for improved biotinylation selectivity A common...exploit the best of both worlds. We generated a plasmid that produces biotin ligase and a protein of interest...and a 6X HisTag for affinity purification (see plasmid map and cartoon below). Including biotin in your...

...packaging plasmids. The cells need three different plasmids to produce AAV: 1) the RepCap plasmid which provides...AAV plasmid standard and AAV transfer plasmids. It’s easy to contaminate a workspace with plasmid, not...going to lie, AAV transfer plasmids are kind of a pain to work with. These plasmids contain ITRs which are...AAV’s protein shell, or capsid; 2) the pHelper plasmid which expresses adenovirus genes which help mediate...mediate AAV viral replication; and 3) the transfer plasmid which encodes a transgene of interest that’s packaged...structures that are prone to deletion from the transfer plasmid. The good news is there’s a few simple ways to ...multiple bacterial cultures of your AAV transfer plasmid at 30 °C instead of 37 °C and then screen for ITR...

...recombined plasmid to overtake full-size plasmids (or for smaller plasmids to overtake larger plasmids, if the...How many unique plasmids does your library contain?), the variance of size of plasmids in your library...lead to issues such as recombination and loss of plasmid diversity, so Addgene strives to distribute aliquots...library (How important is it that every single plasmid in your library is present, and how will you ensure...30 to 200 times as many colonies as individual plasmids in your library. As an example, if you want 100x... your library, and your library contains 5,000 plasmids, your goal for your transformation would be to...libraries often contain thousands, if not millions, of plasmids, they are generally electroporated into electrocompetent...

...organizations choose to share their plasmids under an OpenMTA, which allows plasmids to be used for both research...If you’ve deposited or requested plasmids from Addgene, then you’ve been involved in the material transfer...Even though this process is essential to sharing plasmids around the globe, it can be a bit of a mystery...use agreements. The UBMTA, which is the MTA most plasmids at Addgene are shared under, is well-understood...we are not a party (signatory) to the MTAs for plasmids in our repository. Instead, we facilitate the ...depositor and requestor) organizations. Over the last twenty years, we’ve developed a pretty smooth system...institutes. But they’re not! The vast majority of our plasmids are distributed under the UBMTA, which is widely...

...the transfer plasmid, a plasmid encoding rep and serotype-specific cap genes, and a plasmid encoding adenoviral..., and is routinely tested for mycoplasma contamination using mycoplasma detection kits. Cell line is maintained... tested for mycoplasma contamination. To date, Addgene has never had a case of mycoplasma contamination.... coli. In addition, plasmids are typically prepared using endotoxin-free plasmid purification kits.   ...contamination is minimized by using an endotoxin-free plasmid purification protocol. To minimize the immunogenic...normalization purposes are being used. Quality Control Mycoplasma Our 293T cell line was obtained directly from...offline and discarded. Confirmation of the transfer plasmid Addgene uses a rigorous barcode matching system...

... in one place. Plasmid mapping These tools are for viewing, editing or making plasmid maps, but can also...make plasmid maps. Free for academic users. Serial Cloner: Free desktop-based software for plasmid design...design and mapping. ApE (A plasmid Editor): A free, donation-based plasmid analysis tool including editing...Utah.   Figure 1: A plasmid map created in SnapGene of Blue Flame plasmid pET21a-BirA.   DNA / ...Primer design. Plasmid mapping. DNA sequence analysis. We all have our favorite tools for tackling these...The free SnapGene Viewer is great for looking at plasmid maps and viewing sequencing traces, while the paid... paid version provides more tools for plasmid mapping and design (Figure 1). Benchling: While you might...

...transfer plasmid), the envelope (Env) gene on a separate plasmid (envelope plasmid), and the polymerase ...different plasmids. For a retroviral system this looks like the genetic cargo in one plasmid (the transfer...Gag structural protein on yet a third plasmid (packaging plasmid). Viral production then proceeds by transfection... transfection of all three plasmids into a producer cell line, such as HEK-293T, production of viral particles... and Pol genes are not encoded in the transfer plasmid, the component that physically integrates into ... retroviral packaging components onto distinct plasmids for safety.    Viral cargo-specific safety.... (AAVS1)). For these reasons, AAV is usually classified as a BSL-1 safety level. Adenovirus is similar...

...blog posts on plasmid cloning Resources on Addgene.org Read our protocols for plasmid cloning Find our... from the MRC Laboratory of Molecular Biology. Plasmid cloning is an essential part of any molecular biology... techniques involve the assembly of a circular plasmid in vitro, before transforming it into E. coli for... propagation. However, while not widely known, plasmid assembly can be achieved in vivo using a bacterial...cloning tool was first reported over 25 years ago (Douglas and Howard, 1991, Bubeck et al., 1993). But why...can be encoded in the primer sequences. Multiple plasmids can be included in a single PCR, for example for...linear fragments are transformed into E. coli and plasmid assembly (re-circularization) occurs in vivo. We...

...encoded on separate plasmids and are co-transfected into cells alongside a plasmid encoding the minigenome...four viral genes in the same plasmid (as opposed to four separate plasmids), there are a few reasons arguing...separate (and thus smaller) plasmids. Finally, having four separate plasmids allows researchers to partially... 2 μg minigenome plasmid per well (in a 6 well plate format). However, since plasmid quality and transfection...Minigenome System). From a more technical standpoint, a plasmid with all four genes and necessary control elements...relatively large. Cloning the viral genes in this plasmid would be more difficult compared to if each gene...Technical tips It is important that all of these plasmids (those containing NP, VP35, VP30, L, and the minigenome...

... N-methylpurine DNA glycosylase (MPG), also known as alkyladenine DNA glycosylase (AAG). When adenine ... MPG / AAG N-methylpurine DNA glycosylase / alkyladenine DNA glycosylase  Removes hypoxanthine from DNA...In our last post, we talked about the first base transversion editors: CGBEs, or C → G Base Editors. ...using either fused or endogenous Uracil DNA N-glycosylase (UNG) enzymes. Various other proteins included... (Y = T or C) base editor, which they called glycosylase-based guanine base editor (gGBE) (Tong, Liu, ...Editor Converts C → G by fusing a uracil DNA glycosylase to a CBE; the first type of base transition editor...apyrimidinic (AP) site. UNG / UDG Uracil DNA N-Glycosylase Removes uracil from DNA, creating an abasic site...

...about the amazing years in front of us. So in our last celebratory moment, we wanted to walk through some...on Twitter. In the beginning Let’s take a quick flash back to 2004. “Shrek 2” was the most popular movie...frustrated by the difficulty of accessing published plasmids from other scientists. She, Kenneth Fan, and Benjie...nonprofit that would facilitate the sharing of plasmids between scientists. The service would allow people...Addgene before we were a “labhold” name. Our first plasmid request came on September 8, 2004, for pcDNA-p160MBP... our first major repository milestone — 10,000 plasmids deposited and available — and nearly seven before...possible for scientists to both deposit and request plasmids, was a major focus of our first decade. We did...

...summer or just a bright new plasmid. One area with lots of bright new plasmids is our Fluorescent Biosensors... for AAV packaging.    Find iGECInano plasmids here!   Matlashov ME, et al. Design and Initial Characterization... latest additions to Addgene’s catalog is a new class of sensors for neuropeptides and neurotransmitters...SF-iGluSnFR. Image from Aggarwal 2022.   Find iGluSnFR3 plasmids here! Find viral prep here: pAAV.hSyn.FLEX.iGluSnFR3.... 2022, under CC-BY-NC license.   Find JEDI-2P plasmids here! Find viral preps here: pAAV-EF1a-DIO-JEDI...signaling Receptor tyrosine kinases (RTKs) are a major class of signaling receptors, but the options for measuring...reporter, they observe fluorescence localizes to the plasma membrane only when the RTK is activated and the...

...given protein? Classical methods for studying protein phosphorylation A number of classic methods exist...expression plasmid encoding the ORF to be expressed Finally, the researcher needs a plasmid encoding their...#68306 pCRT7 NT TOPO E17TAG GFP plasmid - Addgene #68295 SupD plasmid – Addgene # 68307 For further technical...Find Synthetic Biology Plasmids Find Bacterial Expression Systems Find Plasmids for Protein Purification... methanogenic archea: these organisms lacked a classic cysteinyl tRNA synthetase, despite abundant incorporation...strain for protein production, and an expression plasmid encoding the open reading frame (ORF) of interest...translation of pSer-incorporated proteins on a single plasmid. This includes 4 copies of a tRNA that specifically...

...names of the plasmids you plan to use Enter the base pair size of those plasmids Enter plasmid concentration...ratios below: 1 Transfer Plasmid : 1 pHelper: 1 RepCap OR 3 Transfer Plasmid : 5 pHelper: 2 RepCap Select...of each plasmid needed per dish or stack. Calculations are based on using 28 μg of total plasmid (Transfer...per 15 cm dish or 2500 μg of plasmids per CF5 stack. Total ug of each plasmid needed for the whole experiment...packaging typically requires transfecting three plasmids at specific molar ratios. To get these ratios ...transfection calculator for AAV packaging Select the plasmid molar ratio you would like to use for your AAV ...experiment Total volume (μL) of plasmid needed for the whole experiment And here’s the equation the calculator...