...organization scheme for multi-part DNA assembly. Simply put, it assembles individual DNA components into larger units...As a plasmid repository, we are no strangers to cloning. If you’re reading this, then chances are you...enzyme and a ligase are used to assemble multiple DNA fragments together in a one-tube reaction. Even though... down into three hierarchical levels: individual DNA parts, transcriptional units, and multigene constructs...transcriptional units. Individual parts are defined DNA sequences with specific functions. Most individual...proteins which interact with other proteins and with DNA, and these complexes regulate the transcription of...systems whose protein products can interact with the DNA that encodes the protein itself or other genes within...

...features was our collection of popular plasmid sequences. These plasmids contain features such as antibiotic...sequence match even when the DNA sequence has changed. Identifying plasmid control elements Coding sequence... our SnapGene powered plasmid maps Learn how we use SnapGene during the plasmid quality control process...out full plasmid sequencing at Addgene Additional Resources on Addgene.org Example plasmid analysis ...available to biomedical researchers manipulating DNA sequences on a daily basis, many found these tools...design was done painstakingly by hand. Records of plasmid construction were often incomplete or nonexistent...know the complete sequences or properties of the DNA molecules they were using. SnapGene was created ...

...ambiguous that the DNA sequencing reaction should be repeated. Guidelines to help with DNA sequencing troubleshooting... sequences! More DNA Sequencing Resources: Tips for Using BLAST to Verify Plasmids Acknowledgements...on BitesizeBio here. As part of my job ensuring plasmid quality at Addgene, I analyze 50-100 sequencing...gel you always include proper controls like uncut DNA and the proper ladder. These controls help you properly...your favorite sequencing facility. When it comes to DNA sequencing the chromatogram is your visual control...the rescue for analyzing and troubleshooting your DNA sequencing results Above (top right corner of this...this post) is an example of a seemingly clean DNA sequence (no Ns in sight). If you never looked at the...

...viral vectors and plasmids with repeating sequences. Repeating sequences within a plasmid can undergo intramolecular...contain the full length plasmid.   Figure 1: The full lentiviral plasmid is expected to be >12 kb...in my samples? Plasmid recombination can affect viral vectors (LTRs and ITRs), plasmids with multiple ...other plasmids that contain repeating elements. Knowing how to amplify recombination-prone plasmids and ... length plasmid. If you find that your bacterial cultures contain a mix of full length plasmid and the...smaller, recombined plasmid around 1.5 kb. When cut with BamHI, the expected linear plasmid is present, but...but the recombined plasmid is still visible. This indicates that the DNA prep contains a mixture of the...

...molecular biologists use to tinker with DNA. Making changes to a DNA sequence can help scientists identify...specific mutations in your plasmids Use REPLACER Mutagenesis to mutate your plasmid Resources on Addgene.... is used to direct the PolI3M-nCas9 complex to a DNA site of interest, which nCas9 nicks and then dissociates...dissociates from. PolI3M then binds the nicked DNA, and extends it from the 3’ end, while its native endonuclease...to a site of interest, where it nicks its target DNA (left). Cas9 dissociates and PolI binds (middle) ...increasing the standard mutation rate seen during DNA replication by 120-fold over background. Due to its... These changes promote nCas9’s dissociation from DNA after nicking and yielded an enhance nCas9 (enCas9...

...restriction enzyme such as BsaI, and a DNA ligase such as T4 DNA ligase. Inserts, either precloned or in...completion of T4 DNA Ligase fidelity studies conducted by Potapov et al. at NEB (1, 2). While T4 DNA Ligase, the... comprehensive profiling of T4 DNA ligase fidelity and bias during DNA end-joining." Nucleic acids research... Gate Assembly is commonly used to assemble 2–10 DNA fragments in a single “one-pot” reaction to form ...junction sequences guided by experimentally-derived DNA Ligase fidelity data. Our work demonstrates that ...antibiotic selectable markers by the destination plasmid. Antibiotic selection allowed for high throughput...accurate assemblies was achieved by the sequencing of plasmids isolated from blue or white colonies. Sequencing...

...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...Library. DNA and cell amounts After deciding on a cell type, you will need to determine how much DNA and how...electroporator uses, etc. The DNA amount can be anywhere from 10 ng to 1 μg of DNA. It often correlates with...Library Type Name Number of Plasmids Variable Region Size DNA per Amplification Amplification...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...

...you can either order a DNA pooled library, which contains a collection of plasmids in a single tube, or ...Transfection of the DNA pooled library and appropriate packaging and envelope plasmids into HEK cells allows...similar to amplifying a plasmid: transform E. coli with the pooled library DNA, allow the bacteria to ...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...library. Addgene’s pooled libraries are typically DNA preps, but we do distribute several of our most popular... prepare the libraries for you.  Starting from plasmids requires amplification, verification, and lentiviral...

...resistance DNA plasmids are traditionally grown in bacteria as a way to produce more of the plasmid, isolate...isolate individual plasmids, or express a gene contained in a plasmid. During the propagation and/or transformation...receive a copy of the plasmid or will lose it during division. These non-plasmid-containing bacteria can...induce cell death by intercalating into DNA and generating DNA breaks. The Sh ble gene provides zeocin...meaning it will induce mutations in the host/plasmid DNA during selection if it’s not completely bound...: Doesn’t work for all bacteria; may cause plasmid/host DNA damage. Choosing an antibiotic resistance ...Over-Agar Antibiotic Plating Plasmids 101 eBook Resources on the Addgene blog Plasmids 101: Antibiotic Resistance...

...enChIP Plasmids? All Hodaka Fujii lab plasmids Sign up to receive email alerts when new plasmids from ...engineered DNA-binding molecules for the first time and named the new technology as engineered DNA-binding... shopping for a wide range of plasmids. The merit of depositing plasmids to Addgene has been spectacular...chromatin immunoprecipitation (iChIP) and engineered DNA-binding molecule-mediated chromatin immunoprecipitation...insertion of recognition sequences of an exogenous DNA-binding protein such as LexA into the target locus...which is the rate-limiting step. When engineered DNA-binding molecule technologies such as TAL proteins... it struck me that direct use of such engineered DNA-binding molecules even enables us to skip the step...

....org Find Cre-Lox Plasmids Find Optogenetics Plasmids Find Chemogenetics Plasmids ...Saccharomyces Cerevisiae 2 Micron Plasmid Attaches Covalently to DNA via a Phosphotyrosyl Linkage.” Molecular...specific (and often cell type specific) control of DNA recombination and gene expression can be advanced...which is named after its ability to invert or ‘flip DNA’, is derived from the yeast S. cerevisiae (Gronostajski...lead to excision, inversion, and translocation of DNA in a similar fashion as between loxP sites (For details...are reversible. Whereas the excision of a piece of DNA flanked by loxP/FRT sites is favored over its reintroduction...the same probability. Continuous inversion of the DNA sequence of interest can lead to poor expression ...

...cuts genomic DNA. The interplay between Cas9 (which causes the breaks) and host cell DNA repair factors...investigations into the release of DNA post-cut. Using substrate DNA labeled on both sides of the break...double stranded donor DNA with other design parameters. Example single stranded donor DNA and a presumptive... Figure 2: Single-stranded donor DNA interaction with the Cas9-DNA duplex. Image from Richardson et al...in uncut target DNA that was accessible to anneal with complementary single stranded DNA. We thus wondered...donor DNA. Chris Richardson is a Postdoctoral Researcher in Jacob Corn’s lab investigating DNA repair...complementary ssDNA molecules in vitro. Figure 1: Cas9 binding to the cleaved DNA duplex. Image...

...integrase-deficient packaging plasmid instead of the standard packaging plasmid. For more details on how to...isolating genomic DNA from transduced cells and quantifying the integrated viral DNA along with a reference...those of the cells they infect. These are typically DNA viruses, such as adeno-associated virus, herpesviruses... transcriptase to convert their RNA genomes into DNA to be integrated into the chromosome of the cell....integrated viral genome is then part of the cellular DNA… forever!  This can be an advantage for researchers...lentiviral genome is distributed across multiple plasmids. For simplicity, we’ll use the second-generation...system as an example.  The lentiviral transfer plasmid encodes your insert of interest, flanked by long...

...Cascade binding and subsequent DNA degradation appears to be conserved. This DNA degradation portion can be...Most Cas effectors have one job — Cas see DNA, Cas chop DNA. Cas3, however, has two jobs, one as a nuclease... kilobases down the strand of DNA, Cascade caught up to it, and then DNA degradation continued. However...and Cas3 begins unraveling the double-stranded DNA (dsDNA) in what is called a reeling mechanism (see next...for details). The newly formed single-stranded DNA (ssDNA) is then pulled through Cas3. After these initial...continually passing the ssDNA past the nuclease domain, Cas3 is able to repeatedly cut the DNA at an efficient...The versatility of CRISPR allows you to play with DNA in a number of ways, from small edits that change...

...the ability to distribute plasmids with the piggyBac™ transposon. These plasmids, when combined with a source... genome fully intact, while, with a standard DNA plasmid transfection, a double-stranded break may occur...utilizing a piggyBac™ transposon in a donor plasmid carrying the DNA edit of interest, along with a site-specific...CRISPR/Cas9) to create a double-stranded DNA break. After DNA cleavage, the piggyBac™ donor is used by...efficiency over transfecting with plasmids in some cell lines, most likely due to DNA toxicity. More importantly...licensed distributor) allow you to quickly transfer a DNA sequence from the transposon vector to one of many...useful resource. While Addgene cannot distribute plasmids with the piggyBac™ transposase itself, please ...

...trained on Addgene plasmid datasets) to predict the lab-of-origin of engineered DNA. Both of these groups...synthetic biology page Search for a plasmid Find Addgene's most popular plasmids ...been coming to the Addgene website in search of plasmids. Now, scientists are beginning to see Addgene ...Addgene as a large data set. Addgene has over 65,000 plasmids in the repository, each verified by sequencing...data to learn about trends in synthetic biology and DNA synthesis. They published their results in a paper...Synthetic biology, in particular, the ability to do DNA synthesis, is evolving so fast that it is a really...variation in GC content between many organisms and DNA synthesis companies often work within a narrow range...

....12137. PubMed. Find the Plasmids at Addgene Kowarz Lab plasmids Izsvak Lab plasmids  ...the gene of interest (blue) in a plasmid backbone. A separate plasmid contains the transposase gene (red...occurs which cuts the DNA.3.) The released transposon can now bind a strand of DNA with a TA dinucleotide...). The original plasmid is empty following the removal of the transposon; the plasmid is then degraded...Transposons are sequences of DNA that can move around in a genome. In a laboratory setting, transposons...transposons combine the advantages of viruses and naked DNA while eliminating some of the drawbacks. Specifically...transposase creates a double strand break in the DNA and allows the tranposon to integrate. Additionally...

...nick-selective DNA ligases like Taq DNA Ligase, HiFi Taq DNA Ligase, and the ATP dependent 9°NTM DNA Ligase are...about it. DNA ligases are enzymes that seal breaks in DNA by joining 5 ́-phosphorylated DNA termini to... several DNA ligases bound to substrate (23, 24). Thermostable DNA ligases, including Taq DNA ligase, ...Blog Learn New Plasmid Cloning Techniques Pushing the Limits of DNA Assembly Plasmid Cloning by PCR ...to 3 ́-OH DNA termini (1-4). In vitro, ligases (notably T4 DNA ligase) are critical reagents for many ...temperatures (37–75 °C). T4 DNA ligase is highly active in nick ligation and DNA end-joining, but will also...gaps. For example, DNA assembly methods, such as Gibson Assembly® and NEBuilder® HiFi DNA Assembly, require...

...highlight a subset of the new plasmids in the repository through our hot plasmids articles. These articles ...recent plasmid deposits and we hope they'll make it easier for you to find and use the plasmids you need...write about a recent plasmid deposit please sign up here. Listen to the Hot Plasmids Podcast!  MarathonRT... 20 nm of a specific DNA sequence in only 1 min. Using these lentiviral plasmids, catalytically inactive...’s lab at Yale University recently deposited a plasmid for expressing a novel reverse transcriptase (RT...biology and enable the conversion of RNA templates to DNA but suffer from several important shortcomings including...). MarathonRT also faithfully transcribes RNA to DNA with low error rates compared to other RTs. MarathonRT...

...evidenced by all the CRISPR publications, press, and plasmids out there, it’s obvious that CRISPR is a ground-breaking...that Cas9 cutting efficiency increases when target DNA strand separation is stabilized (2,3). Stable strand...interactions between the groove and the negatively charged DNA and therefore destabilize basal strand separation...cutting. Figure 1: Strand separation and target DNA binding to the Cas9 nuclease. Stable strand separation...sequence independent interactions between Cas9 and DNA, then they could diminish off-target cutting (4)....between Cas9 and the phophate backbone of the target DNA strand via mutations N497A, R661A, Q695A, and Q926A...compared to eSpCas9(1.1) and SpCas9-HF1. The HypaCas9 plasmids are available from Addgene here! Future possibilities...

...target any sequence of DNA. Second, NgAgo target specificity is dictated by a DNA guide sequence rather...(2016). 3. "NgAgo: Genome-editing by a DNA-guided enzyme." DNA-scissors (blog). Blog website. 4. Hsu, ...be produced from a plasmid in cells. Additionally, in vitro assembly of NgAgo/ssDNA requires incubation...genome editing! Check Out Other Argonaute Plasmids from the Doudna Lab Many thanks to our Guest Blogger ...sequencing on select regions of all distributed plasmids as part of quality control, but does not perform...sharing the stage with much-lauded CRISPR-Cas9 is DNA-guided endonuclease NgAgo! We'll discuss how NgAgo...methods have their own pros and cons (2). The NgAgo DNA guide is inexpensive and can be 5’-phosphorylated...

... Blog Plasmid Cloning by PCR Perform Site Directed Mutagenesis by PCR Catch up on Your Plasmid Background...isolation, insertion and amplification of a recombinant DNA without sequence alteration. Molecular cloning techniques... due to excessive PCR cycles or low fidelity Taq DNA polymerase, the construction of specific sequences...be enough time to completely digest the template DNA. Note—templates must have been isolated from methylation...appropriately sized product, purify and sequence the plasmid to confirm no mutations were introduced during ...PCR or any subsequent steps. Using high fidelity DNA polymerases, I have never had any mutations introduced...Amplification efficiency is highest when using high fidelity DNA polymerases like phusion. An 18-cycle PCR leads to...

...This serves to stabilize DNA-protein interactions for subsequent sheering of DNA by sonification. Though...protocols Browse CRISPR plasmids for isolating a genomic locus Browse FRET plasmids for studying protein ...limitations and makes it easier for you to map protein-DNA interaction with limited biological materials. Problems...Though it helps capture weaker or more transient DNA-protein interactions, the reliance on fixation does increase...CUT&RUN, an enzyme-based protocol for identifying DNA binding patterns that overcomes some of the limitations...collected in a single day. CUT&RUN works by using the DNA cutting activity of a Protein A fused micrococcal...micrococcal nuclease (MNase) to specifically isolate DNA that is bound by a protein of interest. First, nuclei ...

...consider your plasmid's compatibility with one or more other plasmids. Generally speaking, plasmids with the...purify your plasmid DNA: The insert Bacteria tend to maintain fewer copies of plasmids if they contain...elements. The ori is the place where DNA replication begins, enabling a plasmid to reproduce itself as it must...replicons of plasmids are generally different from the those used to replicate the host's chromosomal DNA, but...cell. Figure 1: A plasmid map showing the standard features of a plasmid. So, how do you choose...have only one plasmid type in your cells at a time? Do you want to make a lot of your plasmid? Is the gene...avoid plasmid incompatibility In other words, the best choice of ori depends on how many plasmid copies...

...CRISPR plasmids available at Addgene. We will still periodically focus on specific CRISPR plasmid tools...CRISPR plasmids to tag human transcription factors with GFP. As part of the Encyclopedia of DNA Elements...CRISPR guide Find CRISPR plasmids by function Find recently deposited plasmids ...identified Jennifer Doudna’s lab recently identified Cas14, a family of small DNA-targeting Cas proteins... unlocking) to detect single molecules of RNA or DNA. Now they have developed this further to provide ...Read the paper in Science Find the new SHERLOCK plasmids More precision with dual-nuclease Cas9-Cas9 ...Nature Communications Find the Cas9-Cas9 chimera plasmids A modular toolkit for genome editing in plants...

...stability of modified Ti plasmids during plant infection, as well as plasmid yield during preparation ...selectable marker. Left and right T-DNA borders specify the region of DNA that will be transferred to the ...GreenGate Plasmids Interview: Nicola Patron on Plant Synthetic Biology, MoClo, and More Plasmids 101: Origin...Agrobacterium carries a “tumor-inducing” or Ti plasmid, which allows it to transfer genetic material into...in two species: Agrobacterium and E. coli. Like plasmids used in other systems, binary vectors are cloned...It’s a lot of information to fit into a single plasmid, so most first-generation binary vectors are quite...pKM24KH.To reduce vector size, they shortened the T-DNA borders to 25 bases each. By switching the E. coli...

...comprise a DNA binding domain and an endonuclease domain. What’s distinctive is that the DNA-binding domain... to target a particular DNA sequence? Musunuru: The kit includes 832 plasmids containing prefabricated...effector nuclease (TALEN) kit consisting of 834 plasmids. The researchers showed they could use their TALEN...and can be programmed to bind to essentially any DNA sequence desired. This technology allows you to create...prefabricated pieces that allow you to build a DNA-binding domain to recognize any desired 15 base pair sequence ...the genome in rapid fashion. The two remaining plasmids in the kit have the rest of the protein in place...fluorescent proteins used to sort the cells after the plasmids have been introduced by transfection or electroporation...

...repair DNA. The DNA ends are chewed back to reveal homology, allowing the strands to anneal. DNA synthesis...PITCh plasmid to release the cassette, and an insertion-locus-specific gRNA cleaves genomic DNA. These...Guide Pages Find CRISPR Plasmids for Your Experiments Deposit Your New Plasmid Tools with Us  ...Integration into Target Chromosomes). Using their PITCh plasmids, GFP knock-in cell lines can be created in about...Introduction There are three primary methods for repairing DNA after a double-stranded break. HDR copies the sequence... knock-ins, some labs have used NHEJ for whole plasmid integration; however, since NHEJ is error-prone...additional nucleotides flanking the insertion. If the DNA ends anneal incorrectly, MMEJ may also introduce,...

...disrupting it with an piece of DNA designed in the lab. Specially constructed plasmids can be used to replace ...gene. Here, we'll describe the techniques and the plasmids used to inactivate specific genes in mammalian...mind when choosing a knockout strategy! Knockout plasmids Homologous recombination is a mechanism to accurately...exchanged between two similar or identical molecules of DNA. Gene targeting takes advantage of this natural process...randomly integrated all or large portions of the plasmid. Deliver your construct to your target cells. After...Cre-mediated recombination.   Knockout/Knock-in plasmids Gene targeting methods also make it possible to... and insert GFP into the genome, we’d create a plasmid similar to the one shown below, where the sequence...

...samples at Addgene - DNA sent from depositors, bacterial glycerol stocks, prepped DNA ready for our quality...bacteria with the plasmid can grow. As the bacteria divide, they replicate and retain the plasmid. This allows... Inside Addgene Posts! Learn all about Plasmids in our Plasmids 101 series! ...Every plasmid sample enters Addgene the same way. A package is delivered by a mail courier and then the...dishes, but close to 80% of samples are received as DNA in a microcentrifuge tube. Once a sample enters Addgene...about Developing Lab Management Software    The plasmid's journey begins First, each package is opened and... to hundreds of samples, with an average of 10 plasmids per deposit. Each week an average of 16 deposits...