...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...

...previous posts for our Plasmids 101 series, we examined a number of important plasmid elements – promoters...circular piece of DNA (and is not maintained).  Translocation: If the sites are on separate DNA molecules, ...Resources on Addgene.org Find Cre plasmids Find loxP plasmids  ...for creating (excuse the pun) specific, targeted DNA modifications in transgenic animals, embryonic stem...inserting loxP sites at deliberate locations in a DNA sequence allows for very specific manipulations as...located either on the same or on separate pieces of DNA. Both 13bp repeat sequences on a single loxP site...Cre proteins to form a tetramer. A double-strand DNA break occurs within the core spacer of each loxP ...

...Browse our Plasmids 101 blog posts Read blog posts on plasmid cloning Read about different plasmid elements...or PCR conditions. Purifying DNA fragments Once you’ve generated your DNA fragments, it is always a good...amount of DNA. As a general rule, try to use an excess of insert compared to the backbone plasmid; a starting...mistake or a mistake by the DNA analysis software. Now that you've read our plasmid troubleshooting guide, ...colonies screened might not contain the correct plasmid. There’s a lot that can go wrong! With all the ...cloning project, and hopefully obtain your coveted plasmid with no substantial delays. Design, design, design...spend some time outlining the construction of the plasmid and all the steps you will have to take. Decide...

...of the new plasmids, antibodies, and viral preps in our repository through our Hot Plasmids articles. ...landing sites on opposite DNA strands. When the prime editor releases the DNA, the two 3’ flaps containing...be used for applications beyond cryo-EM.   Find plasmids for MagIC-Cryo-EM here!   Arimura, Y., Konishi...disease phenotype in an animal model. Find PE-eVLP plasmids here! The lab’s other recent deposit builds on...previously-reported technique for precisely integrating large DNA sequences into a genome (Anzalone et al., 2022). ...prime editing and twin prime editing can integrate DNA cargoes of 80–100 bp, PASSIGE can integrate cargoes...over the wild-type enzyme. Find optimized PASSIGE plasmids here!     Figure 3: Schematic of the PASSIGE...

...cells to retain plasmids - if a cell does not inherit plasmid due to improper plasmid partitioning, it...found that if a mini-F plasmid carried a specific 700 bp segment of DNA, the plasmid was propagated better...unstabilized plasmids were retained in less than 10% of cells while plasmids containing the plasmid addiction...death. In the case of plasmids, toxin-antitoxin systems act as “selfish” pieces of DNA whose sole purpose...ensure that plasmids are passed down through generations (Figure 1). These so called “plasmid addiction ...only survive if they retain the plasmid. Since their discovery, plasmid addiction systems have been used...better than plasmids without this DNA. They eventually designated this fragment as the ccdB region (for “...

...containing those overhangs. Entry DNA overhangs may be present in the original plasmid (Option 1) or added using...that flank their DNA fragment in an inverse orientation. This allows for multiple DNA components (promoters...assembly of multiple DNA fragments to create TAL effector nucleases. These plasmids were designed to utilize...Addgene’s plasmids are used with a wide variety of restriction enzyme-based cloning methods. Each method...sequence, they can be customized to direct assembly of DNA fragments. When designed correctly, the recognition...assembly require 20-40 bp of homology at the ends of DNA fragments to specify assembly order, so fragments... and BpiI/BbsI to efficiently assemble up to six DNA fragments at a time. As with all Golden Gate-based...

...subset of the new plasmids and viral preps 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...to contemplate DNA in terms of its sequencing, in vivo studies should always take DNA’s chromatin status... probes target chromatin to visualize DNA, an editor targets DNA (via catalytically inactive dCas9) to...highlights from recent CRISPR plasmids. To find all of the CRISPR plasmids available from Addgene, head...head over to our CRISPR Plasmids and Resources page.  Plasmids for delivering CRISPRi components by a single...inducible degron system for C. elegans New CRISPR plasmids New items from the viral service Epigenetic ...

...lentiviral plasmids include popular envelope and packaging plasmids as well as transfer plasmids. Download...Otherwise, check out our plasmid and DNA backgrounds. Download the blue plasmid background.     You can...the green DNA background.     Since we ❤️ DNA at Addgene, we're presenting you with another DNA background... the plasmids that Addgene offers by visiting our collections page. Here, you can browse plasmids by field...your favorite reagent, scientific technique, or plasmid repository at your next meeting.     If you’re...cute mascots!  Blugene has been helping us make Plasmids 101 videos and needed a break so they're hanging...coronavirus collection page, where you can find plasmid tools to accelerate your COVID-19 research.  Download...

... of DNA through error-prone non-homologous end joining (NHEJ). In targeted DNA knock-in, donor DNA is ...genomes. Such protein-DNA structures presumably reduce the likelihood that donor DNA will interact with ... donor DNA Adenoviral CRISPR/Cas9 genome editing tools can also be used to deliver Cas9 and DNA templates...Gonçalves and Ventura labs’ plasmids, including protocols, check out the plasmids at Addgene: pAdSh.PGK.Cas9...looking for a broader range of CRISPRs plasmid tools, find more plasmids, CRISPR technology guides, FAQs, and...viruses. Adenoviruses are non-enveloped double-strand DNA viruses with a genome of around 36 kb in size, which...Schematic of targeted gene knock-out (left) and targeted DNA knock-in (right) by CRISPR/Cas9. Adenoviral CRISPR...

...sharing plasmids through Addgene is that we provide an added level of confidence in the plasmids we distribute...ensuring plasmid quality is to stay organized. Here at Addgene, we collect data on every plasmid we distribute...location of every plasmid in your collection. Use one of the many freely-available DNA-editing software...cloned into the plasmid) and any other important features that differentiate this plasmid from its predecessor...sequence analysis, plasmid verification, and using NCBI BLAST. Tips for high-throughput plasmid sequencing If...Analyzing and Troubleshooting DNA Sequence Learn Additional Ways to Verify Your Plasmid Learn How Addgene Uses...on the Addgene Website View our Plasmid Reference Pages Get Plasmid Backbone Information from Our Vector...

...bind to a specific DNA sequence via a targeting gRNA. But dCas9 does not cleave the DNA. Much of the research...recognition sequence into the genomic DNA that could then be recognized by a DNA-binding molecule (Hoshino and...chromatin or library of purified DNA for purification of a specific chromatin or DNA fragment (Fujita et al., ... tag expressed on a plasmid, allowing for in vivo biotinylation through a plasmid-based biotin ligase....CRISPR guide Find CRISPR plasmids by function Find recently deposited plasmids ...and Fujii, 2013). They call this method engineered DNA-binding molecule-mediated chromatin immunoprecipitation...immunoprecipitation (enChIP) because any engineered DNA-binding molecule, such as Zinc-finger or TAL proteins, can...

...single base changes in the DNA sequence without completely cutting the DNA. RNA Editors: CRISPR nucleases...Interspaced Short Palindromic Repeats: Sequences of DNA found in bacteria that allow the bacteria to target...bacterial immune system. Genome Editing: Modifying the DNA sequence of an organism’s genome usually for a research...of RNA that direct the CRISPR system to cut other DNA or RNA sequences. Nucleases: Proteins that bind... and are directed by the gRNAs to cut particular DNA or RNA sequences. Cas9 is a very commonly used CRISPR...CRISPR nuclease. Note: Most CRISPR plasmids from Addgene produce either a gRNA, a nuclease, or both....Applications of CRISPR: Cut: CRISPR can be used to cut the DNA sequence. With a repair template, CRISPR can be used...

... to cleave DNA outside of the recognition sequence. This requires that the vectors and DNA fragments contain...developed and fine tuned many different ways to clone DNA fragments which have provided appealing alternatives...scientists are using Gibson assembly to put together DNA fragments. As a fun way to start, I highly recommend...known for allowing easy assembly of multiple linear DNA fragments, but can also be used in basic cloning ... in the figure below. To start, you need to have DNA fragments with regions of homology at their ends,...homology to anneal a polymerase, to fill in the gaps a DNA ligase, to seal the nicks of the annealed and filled-in...can be purchased from a company (e.g. NEB or SGI-DNA), or can be mixed yourself (e.g. see the Miller Lab...

... used for plasmid-based experiments. What are control plasmids? Generally, control plasmids help to ensure...follow an example of a plasmid-based experiment, delineate its requisite control plasmids, and discuss why ... shRNA target correct? Is the purified plasmid DNA viable? Or cytotoxic? How and where did things go wrong...proper control plasmids would address and answer these questions. Types of control plasmids Part of planning... experimental conditions, including how much plasmid DNA to use for any individual or co- transfection...conditions and plasmids should produce a null effect (i.e., no phenomenon is observed). In any plasmid-based experiment... are conducive to the successful delivery of plasmid DNA into the cells, the internal control vector then...

...in molecular biology is DNA→RNA→Protein. To synthesize a particular protein DNA must first be transcribed...and used to manipulate DNA), or in medicine (e.g. insulin). Proteins, unlike DNA which can be relatively... is quite simple; DNA coding for your protein of interest is inserted into a plasmid expression vector...modifications Learn more molecular biology in our Plasmids 101 eBook! Researchers use various techniques ...expression consists of transfecting cells with your DNA template of choice and allowing these cells to transcribe... modifications for proper protein function. Most DNA constructs designed for mammalian expression utilize...from the cellular debris. There are several popular DNA vectors that can be used to produce large amounts...

...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... as a DNA staining dye Article contributed by Jennifer Tsang Figure 4: Double stranded DNA bound... fusions with tTALE to “stain” DNA. TALE proteins can bind specific DNA sequences which brings the attached...attached fluorescent protein to the DNA. They created plasmids for purification of tTALE-eGFP and tTALE-mCherry...need. If you'd ever like to write about a recent plasmid deposit please sign up here.  Human whole genome...alternative to intercalating fluorescent dyes for DNA-protein interaction studies? Kyubong Jo's lab generated...

...that acts as a DNA gyrase poison, locking up DNA gyrase with broken double stranded DNA and ultimately...Addgene Blog Plasmids 101: Common Lab E. coli Strains Plasmids 101: TOPO Cloning Plasmids 101: Golden ...-containing vectors in mind, any plasmid that contains the F plasmid (F’ strains) will also be resistant...Bernard, P., et al. “The F plasmid CcdB protein induces efficient ATP-dependent DNA cleavage by gyrase.” J..., M. “Cell killing by the F plasmid CcdB protein involves poisoning of DNA-topoisomerase II complexes....... The ccdB gene, located on the F sex factor plasmid of E. coli, is part of a toxin-antitoxin system...encoded by the ccd operon, which is responsible for plasmid maintenance during cell division. ccdB codes for...

...crRNA DNA 5' NGG Specific blunt ended DSB in target DNA Cpf1 RuvC-like crRNA DNA 5' TTN Specific...virus, genotype human DNA, identify tumor mutations within cell-free genomic DNA, and detect COVID-19 ...require both a tracrRNA and crRNA to cleave target DNA (Cpf1 requires only a crRNA for target recognition...is that Cas13a binds and cleaves RNA rather than DNA substrates. In terms of structure, Cas13a shares ... Cas9 uses HNH and RuvC domains to cleave target DNA. The HEPN domains within Cas13a are essential for... Specific DSB in target DNA with 5' overhangs Cas13a (C2c2) 2x HEPN crRNA RNA 3' A, U, or C (not required...crRNA to facilitate binding and cleavage of target DNA respectively. LshCas13a, on the other hand, uses ...

... our high-throughput DNA isolation process that yields high-quality isolated DNA samples of sufficient...every isolated plasmid sample and normalize each to fall within a 10 fold range. Plasmids that don't meet...A broader view of the whole plasmid To confirm the sequence of the plasmid, we examine three things: ...expected and do not affect plasmid function, we will update information on the plasmid’s page. Sometimes we’re...’re missing a piece of the plasmid There are regions of some plasmids that are particularly difficult ...some plasmids will not result in one complete, circular assembly. Most of the time, these plasmids are ...bottom of the plasmid page. Viral preps and pooled libraries In addition to regular plasmid sequencing,...

... addition to Taq DNA polymerase, PCR requires free nucleotides (dNTPs), template DNA to amplify from and...unique single stranded DNA primers that bind upstream (5’) and downstream (3’) of the DNA region of interest...crucial for this process as DNA polymerases require an existing strand of DNA to add nucleotides to.  Using...reaction:  Denaturation- To amplify DNA, the two strands of the template DNA first have to be separated. This...primers can anneal to the single stranded DNA template strands. DNA polymerase is also stable at this temperature...specific target of DNA. This amplicon can be used in molecular cloning to create a plasmid of interest, for... of amplified DNA of a known starting concentration. The cycle in which the unknown DNA is detected compared...

... and extract plasmid DNA directly from the colonies. Transform the resultant plasmid DNA into E. coli ... cloning phage DNA can be difficult, in part because phages frequently alter their DNA by adding methyl...fragment library of phage DNA This first step involves digesting phage genomic DNA into 1-3 kb (approximately...involves forcing DNA through a small opening using compressed nitrogen gas; this shears DNA at random locations...If working with DNA that is refractory to enzymatic manipulation, amplifying phage DNA with phi29 polymerase...certain modifications to DNA bases.  Ligate and transform your resultant plasmid libraries into chemically...Step 2: Express random phage DNA fragments in E. coli Now that random DNA fragments have been cloned into...