...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...

...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...

...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...