... Figure 1: A plasmid map created in SnapGene of Blue Flame plasmid pET21a-BirA.   DNA / protein database...Filter DNA tool: Removes non-DNA characters, such as numbers and blank spaces, from the text of a DNA sequence...Primer design. Plasmid mapping. DNA sequence analysis. We all have our favorite tools for tackling these... in one place. Plasmid mapping These tools are for viewing, editing or making plasmid maps, but can also...annotate any DNA sequence. SnapGene Viewer: The free SnapGene Viewer is great for looking at plasmid maps and...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...

...circularized plasmids from plexWell™ NGS data. Once Addgene produces plates of plasmid DNA, they are shipped...synthetic DNA, as well as the advent of extremely fast, cheap and accurate sequencing (“reading”) of DNA, have...large numbers of plasmids. plexWell™ technology overview For any NGS experiment, the DNA sample being sequenced...of samples are subjected to a DNA-barcoding step that uniquely labels DNA from each well of a 96-well plate...numbers of individual input DNA samples (e.g. a collection of 96 plasmids on a plate) will typically have... not vary by DNA input amount. plexWell™ + Addgene = high quality NGS-assembled plasmids Because of the...additional information about the presence of non-plasmid DNA or heterogeneous and/or mixed clones present ...

...Deletions in a Plasmids Learn How to Verify Your Plasmid Resources on Addgene.org Find More Plasmid Cloning...enzyme recognizes the DNA sequence 5´-(C/T)CCTT-3' and digests double stranded DNA at this sequence. The...different DNA strand comes along, it can attack this covalent bond thus joining the two DNA strands and...Topoisomerase based cloning (TOPO cloning) is a DNA cloning method that does not use restriction enzymes...comes from a topoisomerase I-linearized backbone. DNA topoisomerase I (depicted as a green cloud) functions...as a ligase by cleaving and rejoining supercoiled DNA ends to facilitate replication. The TOPO technique...stored as a covalent bond between the cleaved 3′ DNA strand and a tyrosyl residue of topoisomerase I (...

...27136078. Find plasmids from this publication at Addgene. 2. Swarts, Daan C., et al.  “DNA-guided DNA interference...GC-rich regions. DNA guides NgAgo uses 5’ phosphorylated DNA guides (so called “gDNAs”) rather than the...that this protein could cleave DNA in vitro, directed by phosphorylated DNA guides. Unfortunately, Swarts...identified NgAgo and showed that it can cleave plasmid and genomic DNA in a variety of mammalian cell lines, with... guide DNA and cannot switch guides. While this fidelity would prevent other small nuclear DNAs from binding...archaeon Pyrococcus furiosus is a DNA-guided nuclease that targets cognate DNA.” Nucleic Acids Res. 43(10) ...going gaga over the newest gene-editing protein - a DNA-cleaving Argonaute from Natronobacterium gregoryi...

...Universality All types of DNA fragments may be cloned: PCR fragments, cDNA or Genomic DNA and is available for...clone with the DNA of interest flanked by attB sites is generated. As in the BP reaction, a DNA fragment containing... the entry clone with your DNA sequence of interest, you can move this DNA fragment across any expression...multiple DNA fragments into many vectors at once in the same tube. You can clone up to 4 DNA fragments...kit, and MultiSite Gateway plasmids. Constant reading frame When you move a DNA fragment from one Gateway...to find Gateway plasmids for your organisms of interest: Find Gateway cloning plasmids here! References...popular as a rapid and highly efficient way to move DNA sequences into multiple vector systems. With the ...

...Addgene blog:  Plasmids 101: What is a plasmid? Plasmids 101: Origin of ReplicationPlasmids 101: PCR...process known as plasmid multimerization. Plasmid Multimerization Early studies on plasmid topology recorded...to Detect Plasmid Multimers Run an undigested plasmid on agarose gel Running undigested DNA on a gel as...ligate the plasmid, and re-transform the ligated plasmid to recover the monomeric form of the plasmid. Have...restriction digests on plasmids at Addgene, we sometimes observe something odd in our uncut DNA control: a band...expected, given the size of the plasmid. This is seen only in the uncut DNA; the rest of the digest appears... monomer of Plasmid 12259: pMD2.G (depicted on the left) is ~5.8 kb in length. The plasmid can exist on...

...orientations, a SSR will invert the DNA sequence between the sites. If a DNA sequence is floxed in the same...end of the DNA fragment (Figure 1, middle). A second recombination event then excises the DNA between the...In a previous post from our Plasmids 101 series, we learned how the Cre-loxP recombination system can... recombinase to invert, translocate, or excise a DNA fragment. The availability of both wild-type and ...site-specific recombinases (SSRs) Cre and FLP. SSRs bind DNA at target sites to induce site specific recombination...binds loxP sites, while FLP binds FRT sites. When a DNA sequence is flanked by target sites (floxed) in opposing...same orientation, the SSR will excise the DNA fragment. By manipulating the number, orientation, and type...

...flexibility to modify the E. coli chromosome, plasmid DNA or BAC DNA.  To use the lambda red recombineering...your target DNA, you first electroporate a linear donor DNA substrate (either dsDNA or ssDNA – see below... system. MMR’s job is to correct DNA mismatches that occur during DNA replication. There are two ways ...you use depends on what type of DNA you want to edit; however, BAC DNA can be modified with any of the...background via P1 transduction. Plasmid Expressing lambda red genes from a plasmid allows for a mobile recombineering... what type of DNA is being targeted for modification: the E. coli chromosome, a plasmid, or a BAC.    ...   Substrate DNA Design and Generation Whether you use a linear dsDNA or ssDNA substrate depends ...

... involving two-plasmid or single-plasmid systems (Vo et al., 2020). In the two-plasmid system, an effector...TnsA, TnsB, TnsC). The donor DNA (aka mini-transposon), which contains the DNA cargo of interest flanked ...complexes survey the genome and bind target DNA sequences using RNA-DNA base pairing. Separately, TnsABC forms...components for RNA-guided DNA integration, Type V-K systems frequently produce off-target DNA insertions and intermediate...CRISPR array Clone desired DNA cargo into the mini-transposon Transform plasmid(s) into cells and plate ...Sternberg Lab at Columbia University Medical Center. DNA transposons are ubiquitous genetic elements capable...Sleeping Beauty). Despite their diverse utility, DNA transposons have been hindered by their limited programmability...

...the pool down to plasmids expressing variants with optimal function. High-throughput DNA sequencing is then...homologous DNA fragments in a single-tube reaction. For mutational scanning experiments, libraries of DNA fragments...recognizes uracils that are in DNA and ExoIII recognizes nicked duplexed DNA. This results in the final product...restriction site, but nick opposite strands of DNA. Treating the plasmid with ExoIII and ExoI then fully degrades... Can I use my favorite plasmid with this protocol? Maybe. Not all plasmids will work for the one-pot...strands of the plasmid will be nicked during the first BbvCI digest and the whole plasmid will be degraded...those in the plasmid. Again, if they are in the opposite direction, both strands of the plasmid will be nicked...

...mutagenesis of Plasmids. A) Generation of mutagenized plasmids. PCR primers (green) amplify the plasmid template...with the nuclease-resistant nicked plasmid (the PCR product). Plasmids are isolated from the resulting colonies...the final circular plasmid product pictured here. B) Screening of recovered plasmids for the mutation of...Smaller plasmids (~3 kb) are generally more efficiently amplified than larger constructs, but plasmids as ...DpnI) to eliminate the parent plasmid from the PCR products, the plasmid template should be isolated from...mutagenesis. Nuclease To remove the template DNA (unmodified plasmid) a restriction digest with DpnI is used...efficiently patch up the DNA. The resistance marker from the parental plasmid provides a mean for selecting...

...covalent modifications to DNA or histones, the proteins that help package DNA, were associated with gene...initiates cytosine demethylation of DNA. However, several proteins in the DNA oxidation and repair pathways ...site-specific DNA methylation. Biology Open 5:866–874 . https://doi.org/10.1242/bio.019067 Find plasmids from ...targeted DNA demethylation. Cell Discovery 2: . https://doi.org/10.1038/celldisc.2016.9 Find plasmids from...demethylation in mammalian cells. This plasmid is used with pcDNA3.1-MS2-Tet1-CD to decrease methylation ...BioRender.com. DNA Methyltransferase MQ1 Margaret Goodell's lab has deposited pcDNA3.1-dCas9-MQ1(Q147L...gene expression or silencing. To alter DNA modifications, researchers used blunt tools like histone deacetylases...

...remove parental DNA (37 °C for 15 mins). This enzyme specifically cuts methylated DNA, so it will selectively...primer design.  Use minimal template DNA Use 1 ng of PCR template DNA to minimize false positive colonies...after PCR means successful plasmid assembly (if primer design is correct). Some DNA sequences are not easy...possible Some plasmids just won’t amplify! Examples of this are the GC-rich ITRs of AAV genomic DNA encoding...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...

...CRISPR plasmids available at Addgene. We will still periodically focus on specific CRISPR plasmid tools...optogenetic repressor plasmids Cytosine base editors with minimized off-target DNA editing The original...independent off-target DNA editing, ~5–50-fold lower levels of Cas9-dependent off-target DNA editing, and maintain...paper in Scientific Reports Find the plasmids Enhanced CRISPR-based DNA demethylation Figure 2: ...CRISPR guide Find CRISPR plasmids by function Find recently deposited plasmids ...gRNA for human pluripotent stem cells CRISPR-based DNA demethylation Optogenetic repressors of gene expression...induces a low frequency of genome-wide off-target DNA editing. Using several rapid and cost-effective screening...

...primer for DNA repair synthesis. The ssDNA strand then displaces one strand of the homologous DNA donor and.... The most dangerous type of DNA lesion results from breakage of both DNA strands - a double-strand break...error-free provided the DNA template used for repair is identical to the original DNA sequence at the DSB,...mutations into the damaged DNA if these modifications exist in the template DNA. There are several unique...central steps: The 5’ DNA end of the break is resected by nucleases to create a ssDNA 3’ overhang. This will...longer than 200 bases can be difficult, thus dsDNA plasmids are generally preferred for large insertions...toxic to cells than their plasmid counterparts. Another consideration for dsDNA templates is their ability...

...More: Plasmids 101: Antibiotic Resistance Genes Plasmids 101: Origin of Replication All Plasmids 101 Articles...Although plasmids do not naturally exist in mammals, scientists can still reap the benefits of plasmid-based...introduction of DNA into bacterial cells); however, the techniques and reagents vary. Plasmid transfection...and the resultant cells can either express the plasmid DNA transiently (similar to bacteria) or incorporate...sufficient for the transfected plasmid to be expressed transiently. Since the DNA introduced in the transfection...nuclear genome, in the absence of plasmid replication, the foreign DNA will be degraded or diluted over...their genomes. Unlike plasmids used for expression in yeast and bacteria, plasmids used for stable transfections...

...although higher DNA concentrations are needed for this method to be successful. Low DNA concentration? .... They just need enough complementary ssDNA to hold the plasmid together, even if there are gaps or flaps...SLIC is a cheap, standardized, and rapid multi-part DNA assembly method. Read on to learn how to use it in...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... let them do the repair work. The key is T4 DNA polymerase, which has 3′→5′ exonuclease activity. T4’s...

...referring to any DNA within the context of a nucleus as “linear” is a misnomer. The DNA packed into our...Chromatin-related genes with plasmids available from Addgene The links below pull up lists of plasmids containing the...containing lists of plasmids from the indicated publications. You can request all of these plasmids directly from... looked at a diagram depicting transcription, or DNA repair, or replication, or any number of CRISPR applications...every diagram portraying some aspect of eukaryotic DNA would become busy and potentially detract from the... scientists who not only remember to think about DNA in this context, but also specifically study it. ... loops, chromosomal domains, or how stretches of DNA on different chromosomes interact with each other...

...site, and the deaminase induces the DNA base change without a DNA double-strand break. But base editors...the DNA. ABEs are somewhat slow and need a strong Cas9 domain to “hold on” long enough to the DNA to induce...following components: A plasmid in the host bacterium expressing dCas9 A plasmid in the host bacterium ...efficient in on-target DNA editing as ABE8e while maintaining the lower off-target DNA and off-target RNA ...Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage. Nature 551:464–471 . https://doi....editing capabilities. Base editors make specific DNA base changes and consist of a catalytically impaired...impaired Cas protein (dCas or Cas nickase) fused to a DNA-modifying enzyme, in this case a deaminase. Base ...

...addition to human error, fragments of DNA from the helper plasmids or the cell genome can be packaged inside...sequence all the DNA packaged inside the viral particles (Figure 1). Briefly, packaged DNA is extracted from...find DNA from the packaging cell genome, bacterial genomes, cloning vectors, and helper plasmids (Chadeuf...used to identify DNA contaminants in AAV preparations and provide information on these DNA species with exquisite... tube is mislabeled or someone grabs the wrong plasmid? What if bacteria snuck into the prep during the...determine the identity and serotype of the packaged DNA and look for potential contaminants. The analysis is ...) are aligned to the reference sequence of the plasmid used to create the viral prep. In a clean viral...