...Broad GPP genome-wide Brunello 73179 (1 plasmid) 73178 (2 plasmid) Knockout Human Doench and Root 3rd 4 ...76,441 Broad GPP genome-wide Brie 73632 (1 plasmid) 73633 (2 plasmid) Knockout Mouse Doench and Root 3rd 4 ...GPP kinome Brunello 75314, 75315 (1 plasmid) 75312, 75313 (2 plasmid) Knockout Human Doench and Root 3rd... 3,052 Broad GPP kinome Brie 75317 (1 plasmid) 75316 (2 plasmid) Knockout Mouse Doench and Root 3rd 4 ...20,051 Human GeCKO v2 1000000048 (1 plasmid) 1000000049 (2 plasmid) Knockout Human Zhang 3rd 6 123,411...185,836 Mouse GeCKO v2 1000000052 (1 plasmid) 1000000053 (2 plasmid) Knockout Mouse Zhang 3rd 6 130,209... - 3 plasmid system 1000000075 (Puromycin) Activation Mouse Zhang 3rd 3 69,716 SAM v2 - 2 plasmid system...

...restriction digest, and then plasmid sequencing if you can. To quote Doench, “Whole plasmid sequencing is just ...correctly banded clones for plasmid sequencing and found 98% of the plasmids were perfectly correct. Because... Mammalian Plasmid pRDA_791 (contains only orio and ampR) Drosophilia Plasmid pRDB_051 (for...the assembled plasmid. It shows you each component, with the ID of the component plasmid, component name...the component plasmids on the Addgene website. Step 4: Naming your assembled plasmid and generating...resources from Addgene Plasmids 101: Modular Cloning Addgene's CRISPR Guide Plasmids 101: Golden Gate Cloning...Mammalian or drosophila) Delivery type (lentivirus, AAV, transposon, Plasmid) Destination vector (dropdown options...

...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...services Addgene Biosafety Guide Addgene Viral Plasmids Which Type of Virus Should I Use? Resources...

...producing my lentiviral plasmids? Due to the long terminal repeats found in lentiviral plasmids, we recommend...into a lentiviral plasmid containing only one restriction site? If a lentiviral transfer plasmid contains...integration. Packaging plasmid(s) Envelope plasmid Addgene’s packaging and envelope plasmids are generalized ...and optimize is the transfer plasmid. 2nd generation lentiviral plasmids utilize the viral LTR promoter...third, separate, envelope plasmid. All 2nd generation lentiviral transfer plasmids must be used with a 2nd... below. Third Generation Lentiviral Plasmids 2nd vs 3rd Generation Lentiviral Systems Feature 2nd Generation...packaging systems Packaging Plasmid All on one plasmid: Gag, Pol, Rev, Tat Two plasmids: one encoding Gag and...

... Biosensors Luciferase Plasmids Subcellular Localization Optogenetics Plasmids Viral Service: AAV Biosensors...Kit from the Cohen laboratory includes 19 lentiviral plasmids for a variety of fluorescent sensors to monitor...suggestions for other plasmids that should be added to this list? Fill out our Suggest a Plasmid form or e-mail...Collection of plasmids encoding fluorescent biosensors to measure biomolecules or genes via FRET or other... Plasmid Collections Fluorescent Proteins Fluorescent Biosensors Fluorescent Proteins: Biosensors...our catalog . Use the article links to find the plasmids associated with the article. We also offer ready-to-use...ready-to-use AAV preparations of select plasmids; these are noted with the viral service icon . Metal Ions...

...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...

.... With others, notably pooled lentiviral plasmid libraries, the plasmids must first be used to make virus... Science Guides Guide to Plasmid Pooled Libraries Plasmid Pooled Libraries Jump To: Pooled Library...are single preparations of many different plasmids. Plasmids within a given library have the same backbone...libraries, each plasmid contains a unique cDNA. In shRNA or gRNA libraries, each plasmid contains a unique...infected cell receives only one plasmid. To make sure that every plasmid is adequately represented in the... given plasmid, think of each cell carrying it as being a biological replicate for that plasmid. Library...the overall library. Barcoding libraries contain plasmids with unique, semi-random sequences that can be...

... LTRs, differ. As a result, lentiviral and γ-retroviral packaging plasmids are not interchangeable. General...envelope plasmids, such as VSV-G, however, may be used across both systems. Find retroviral plasmids available... 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...different “generations” associated with lentiviral transfer plasmids are determined by the presence/absence...do many γ-retroviral plasmids have similar names? The nomenclature of the plasmid backbone is meant to...selection. Return to Top Glossary Plasmid Type Element On Same Plasmid as Transgene? Purpose Envelope VSVG...

... educational tools Playing BUILD YOUR PLASMID BUILD YOUR PLASMID is a game where you need to apply basic... videos to teach basic lab techniques Plasmid Land: The plasmid cloning game ... post, BUILD YOUR PLASMID has been developed to teach gamma-retrovirus or lentivirus-based ex-vivo gene... gene and Selection Markers) and one “Transfer Plasmid” game board. Once they agreed on each element to...minutes to develop a strategy and build a transfer plasmid that could be used to genetically manipulate human...disease someday! Accessing and modifying BUILD YOUR PLASMID You can find rules, suggestions, and more details...different education levels. If you modify BUILD YOUR PLASMID or generate games like it, please freely share ...

...Download our Plasmids 101 eBook to get all the background you need to start working with plasmids! What are...Techniques Read all of our Plasmids 101 Posts Resources on Addgene.org Browse our Plasmid Reference Pages Just...Plasmids designed to express genes in a given host cell type are generally broken down into two broad...based on the functional elements they contain. Plasmid DNA in both prokaryotic and eukaryotic systems ...Rho-dependent terminators are not usually employed in plasmid-based expression systems, so these will not be ...the end.  Nearly all common bacterial expression plasmids use Rho-independent terminators, which include...termination with polyadenlyation. Mammalian expression plasmids are primarily used to create mRNA and the commonly...

...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...perform the transduction with: either retrovirus, lentivirus, adenovirus, or AAV. Then, your desired DNA to... some viruses integrate into the host genome (lentivirus and retrovirus) while others are non-integrating...Addgene.org Generating Stable Cell Lines with Lentivirus Virus Handling and Storage Addgene Viral Services...

...Ready-to-Use AAV Find Ready-to-Use Lentivirus Browse Viral Plasmids ... tube is mislabeled or someone grabs the wrong plasmid? What if bacteria snuck into the prep during the... human error, fragments of DNA from the helper plasmids or the cell genome can be packaged inside the ...) are aligned to the reference sequence of the plasmid used to create the viral prep. In a clean viral...bacterial genomes, cloning vectors, and helper plasmids (Chadeuf et al., 2005, Wright et al., 2011). In...collection database, and it may also be aligned to plasmids in our inventory containing the suspected contaminating... the best match for the contaminant is Addgene plasmid #44362. Method validation, limitations, and future...

...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...blog Parts of the AAV Packaging PlasmidParts of the AAV Transfer PlasmidAAV Variables That Matter AAV protocols...production process.  Helper-free production Unlike lentivirus, AAV does not possess the ability to replicate...

...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... (~5 kB payload for AAV and ~8 kB payload for lentivirus). Large cargos, such as the ~11 kB cDNA for dystrophin...

... If there’s a plasmid or AAV tool that you can’t find, you can always suggest a plasmid to us, request...want to get into the field. Find Optogenetics Plasmids for Use in Neuroscience Research Neuroscience ...400 who signed up for our newsletter and a free plasmid) were very excited about our new viral services...Service Find Retrograde AAV Browse Optogenetics Plasmids ...ready-to-use adeno associated virus (AAV) and lentivirus are now available from Addgene. While super useful...

...complicated procedures. Plasmid Cloning Protocols for constructing and analyzing your plasmids. Virus Protocols...Amplify CRISPR pooled-plasmid libraries Diagnostic Restriction Digest Verify a plasmid using restriction ...Transfection Introduce plasmid DNA to mammalian cells Lentivirus Production Produce lentivirus with a polyethyenimine... that can be used for basic molecular biology, plasmid cloning, and titering and testing your viral preparations...Bacterial Glycerol Stocks Store bacterial strains or plasmids for long term use Watch the Video! DNA Purification...spectrophotometer Restriction Digests Cut and analyze plasmid DNA with restriction enzymes Agarose Gel Electrophoresis... the Video! Sequence Analysis Verify important plasmid features using sequence analysis RNA Extraction...

...high efficiency Lentiviral transduction Cas9 and gRNA can be present in a single lentiviral transfer vector...Packaging and envelope plasmids provide the necessary components to make lentiviral particles (for details...Additionally, many plasmids containing validated gRNAs are now available through Addgene. These plasmids contain ...particular model organism. There may not be a perfect plasmid for your specific application, and in such cases...different activators exist, including the multi-plasmid SAM system. Select Expression System To use CRISPR...to generate stable cell lines (for details, see Plasmids 101: Mammalian Vectors ) Transient or stable expression...Read more about how to design your gRNA . Browse Plasmids: gRNAs Synthesize and clone desired gRNAs Once...

...on the Addgene Blog Plasmids for endogenous gene tagging in human cells Plasmids 101: Protein Tags Fluorescent...Browse CRISPR plasmids Check out our CRISPR guide pages Find ready-to-use Cas9 lentivirus ...from many pitfalls: they require construction of plasmid vectors and/or rely on cumbersome in vitro synthesis...directly purchased and there is no need to construct plasmid vectors or synthesize/purify IVT sgRNA in the lab...

...tRNA-gRNA arrays, which is scalable and efficient in plasmid and viral vectors. In applications when repetitive...manual and online design examples of using Addgene plasmids hCtRNA_FT (#186715) and hCtRNA_VT (#186716).   ...deliveries via adeno-associated virus (AAV) and lentivirus, demonstrating simultaneous editing of genes ...

...genetically encoded, and luckily, we have pre-designed plasmids for these systems! Ion sensors One common biosensor...we’ve got you covered with packaging plasmids! References and Resources References  Bhalla N., Jolly ...ready to deliver your sensor, whether you choose lentivirus, gamma retrovirus, or AAV as your vehicle, we...