...cloning plasmids, expression plasmids, gene knock-down plasmids, reporter plasmids, viral plasmids, and ..., modify, or construct a plasmid. A plasmid is a small circular piece of DNA found in bacterial cells,... of scientists and plasmid lovers - serves as an introduction to plasmids. Plasmids 101 will provide you...  What is a plasmid? At their most basic level, plasmids are small circular pieces of DNA that replicate...that make up a plasmid and why each is important. Our “Plasmids 101” series designed to educate all levels...contrast, plasmids utilized in the lab are usually artificial and designed to introduce foreign DNA into another...cloning site. The ease of modifying plasmids and the ability of plasmids to self-replicate within a cell ...

...levels of protein, not plasmid DNA. If you need large amounts of DNA from a plasmid in a protein expression...to an improved plasmid DNA yield. Culture conditions play a critical role in plasmid DNA yield. Different...co-purify with your plasmid DNA and lead to degradation of your prep. Many plasmid prep kits recommend...more cells = more plasmid. Plasmids can vary in copy number, the number of plasmid copies supported within...sub-culture, and prep with a DNA prep kit or your lab's favorite in-house protocol. DNA yields for this procedure...excess of 100 ng/ul, more than enough DNA to verify your plasmid via sequencing or restriction digest....where your plasmid yield is sub-optimal? If you are consistently getting sub-optimal plasmid yields from...

...elute and store your plasmid DNA Learn about the different grades of plasmid DNA preps Resources on Addgene.org...signs of DNA in your final tube after purification. How do you know if you actually have DNA in your tube...of the DNA sample.  These absorbance measures give you an idea of the concentration of your DNA prep and...sensitivity at low concentrations of DNA and it can’t distinguish between DNA and RNA. See our protocol about...UV absorbance to quantify DNA Fluorescence dyes Another way to quantify DNA would be to use fluorescent...way to quantify DNA, you can use the agarose gel method to not only find out how much DNA you have, but ...fluorometer to quantify DNA this way. First, start by pouring your gel containing a DNA intercalating dye (...

... ways: Transformation grade DNA Cloning grade DNA Transfection grade DNA Figure 1: Transformation...process of inserting exogenous DNA in a microorganism. The amount of plasmid DNA that is necessary for bacterial... into a plasmid backbone. Transfection grade DNA Figure 3: Need to introduce plasmids into mammalian...utilize your plasmid DNA, so that you can decide what will be the optimal type of plasmid preparation ...Not all plasmid preps are the same. Before purifying a plasmid from a bacterial culture, it is important... of DNA you need, and at which level of purity. Based on these premises we can classify a plasmid preparation...transformation grade plasmids into a bacterial cell. Transformation grade DNA  Transformation is the...

...fragmenting plasmid DNA, PCR product, or chromosome. One of the main causes of degradation during plasmid DNA ...want to use the plasmid DNA can help you choose the best way to store and elute the DNA. References Murakami... to purify DNA without a kit Read our blog posts about plasmid cloning Browse our Plasmids 101 blog posts...experiments you can effectively use the DNA. pH and DNases affect DNA stability Figure 1: So many choices...responsible for the sugar backbone of the DNA helix or even changes the DNA sequence through a mechanism called...adenine or guanine) from the DNA. These apurinic sites if in double stranded DNA are repaired by base excision...degradation and which experiments you can use your plasmid.  DNA elution choices: TE, Tris buffer, or water ...

... you don't have the full plasmid sequence. Once you have purified plasmid DNA, this method can be done...Always run control uncut DNA to ensure your enzymes are working. When uncut plasmid DNA is isolated and run...the plasmid based on the predicted sizes and organization of different features within the plasmid. Restriction...steps: 1) incubating your DNA with restriction enzymes which cleave the DNA molecules at specific sites...example plasmid on the right has a total size of 7.3kb, including a 1.2 kb insert. The plasmid was digested... as a reference band. The uncut DNA (lane 2) shows 3 possible plasmid conformations, with relaxed and ... pouring it. EtBr binds to the DNA and allows you to visualize the DNA under ultraviolet (UV) light. Don...

...and genomic DNA to precipitate. Plasmid DNA is free in solution. Proteins and genomic DNA are removed ...agarose gel slices Product: plasmids or DNA fragments The plasmid purification and DNA gel extraction method...modifications depending on the type of DNA you’re purifying (plasmid, genomic, or DNA fragments from agarose).    ...alkaline lysis plasmid miniprep Starting material: 2 mL bacterial culture Product: plasmid DNA This kit-free...solubilizes both DNA and agarose, and 2) it helps DNA stick to the glass. After binding DNA, the glass particles... Read our Plasmids 101 blog series Find other plasmid protocols and tips Browse our plasmid cloning blog...column, stop to consider some ways to purify DNA without a kit. DNA purification kits have advantages: they...

...weight genomic DNA; filter the remaining plasmid DNA through a column that binds the plasmid DNA and lets other... your insert (or donor plasmid) and plasmid backbone. Because you lose some DNA during the gel purification...restriction enzymes to cut open a plasmid (backbone) and insert a linear fragment of DNA (insert) that has been ...restriction enzymes. An enzyme, DNA ligase, then covalently binds the plasmid to the new fragment thereby ...restriction cloning process. Both the plasmid (blue, backbone) and the DNA sequence of interest (green, insert...insert and 1μg of plasmid backbone. It is also critical that as much of the backbone plasmid as possible be... of the backbone plasmid (see below). You should treat your digested backbone plasmid with a phosphatase...

...general, these plasmids tend to be low copy number. Conversely, plasmids that can initiate DNA replication...growth against sustainability. Plasmid DNA replication and regulation Plasmids must also strike a balance:...nearby AT-rich regions of DNA. In a test tube, the DNA helix can be separated by DnaA alone, as long as there...replication of the host cell's chromosomal DNA.  Plasmids are said to be under stringent control when...replication mechanism of plasmids under stringent control, such as pSC101. DNA replication and regulation...at an example plasmid, let's review how E. coli normally replicate their chromosomal DNA. Chromosomal ... ori. In the elongation phase, a DNA polymerase synthesizes a new DNA strand at each replication fork....

... successfully takes up plasmid DNA. Among those that do, some now have plasmids that contain your gene...of interest, get it into your plasmid backbone, and zap the mixture of DNA into cells. Unfortunately, not...antibiotic. For example, if the DNA you’re inserting into the plasmid backbone contains a kanamycin resistance...been transformed with the plasmid backbone (with or without your inserted DNA) and further screening will...gene is found on the original plasmid and either the insertion of a DNA fragment within the gene or loss...including ccdB on its destination plasmids. In the destination plasmid, the ccdB gene is flanked by recombination...selections in plasmid cloning. Additional resources on the Addgene blog Read our Plasmids 101 blog posts...

... you are amplifying from plasmid DNA (rather than from genomic DNA or a cDNA library), roughly 18-21bp... on All Things Plasmid with Our Plasmids 101 Series Get Tips on Verifying Your Plasmid Resources on Addgene.org...Check out Our Plasmid Reference Pages Deposit Your Completed Plasmids Find the Perfect Plasmid for Your Research...and NotI to ligate our cDNA into the recipient plasmid. Remember to insert your DNA in the correct orientation... piece of DNA and at the same time adding restriction sites to the ends of that piece of DNA so that it...recipient plasmid (usually in the Multiple Cloning Site (MCS)), but do not cut elsewhere on the plasmid Bonus...essential that you verify your final plasmid by sanger sequencing. DNA replication by PCR has error rates...

...fragments of DNA to create a synthetically designed DNA sequence. There are multiple methods of DNA assembly... scarless plasmid inserts.  Want to Learn More about DNA Assembly? Check Out Our Plasmids 101 eBook!...New England Biolabs. What is DNA assembly? In the context of cloning, DNA assembly refers to a method ... NEBuilder® HiFi DNA Assembly. Gibson Assembly allows the production of scarless DNA constructs using ...shuffling of DNA regions. Golden Gate Assembly also allows the creation of standardized DNA constructs,...homology) cut from plasmids via restriction enzyme digestion, and used directly in DNA assembly will still...as easy as obtaining a plasmid with the gene of interest (search Addgene plasmids by gene here), designing...

...adjacent lysed bacteria and can include plasmid DNA or fragmented DNA released into the environment. Various...don't exactly "eat" plasmids as depicted in this comic, when a bacterium uptakes a plasmid, the genes on the... the plasmid can give them different phenotypes. For example, this plasmid encodes a green fluorescent...bacterial DNA can be accidentally packaged into the newly created phages. Transfer of this DNA to another...Transferred DNA once inside the infected bacterium can either exist as transient extrachromosomal DNA, like ...commonly use phagemids, a DNA cloning vector that contains both bacteriophage and plasmid properties. The phagemids... Read all of our Plasmids 101 blog posts Check out all of our blog posts on plasmid cloning Resources...

...to Elute and Store Your Plasmid DNA? by Paolo Colombi Should I store my plasmid in TE, Tris, or water?... a subset of these tools for plasmid mapping, DNA/protein databases, DNA sequence manipulation, and primer...new cloning grade DNA service. We've had 11 blog posts on COVID-19 featuring plasmid tools from depositors...launched the cloning grade DNA service. This service features 210 plasmids: empty backbones, fluorescent... tags, SARS-CoV-2 plasmids, etc. It is our hope that by providing cloning grade DNA to you, we'd save ... development virtually. 6. Quantifying DNA? Here are Five DNA Quantification Methods to Consider by ... dominates DNA quantification discussions, but what about the other methods to quantify DNA? Here we cover...

...Unmethylated DNA not degraded, cell still can methylate DNA   hsdS(rk-,mk-) Unmethylated DNA not degraded...cytosine DNA Allows for uptake of foreign (methylated) DNA recA, recA1, recA13 Mutation in a DNA-dependent...recombination and general DNA repair Reduces plasmid recombination, increases plasmid stability recBCD Exonuclease... routine cloning, cloning unstable DNA, preparing unmethylated DNA, and more. Many mutations that make...improvements such as those that increase plasmid yield and/or DNA quality. Table 1 below outlines a few ... Consequence dam DNA adenine methylase mutation (GATC) Preparing unmethylated DNA, important when trying...ClaI or XbaI) dcm DNA cytosine methylase mutation (CCWGG) Preparing unmethylated DNA, important when trying...

...using plasmid tools. Details about the cloning grade DNA beta project We will ship you  2 µg of DNA in ... after ordering a plasmid you didn't have to grow the bacteria and prep the plasmid before you begin your...grade DNA service and is ready to use out of the box. We estimate that providing cloning grade DNA could...discovery.” Here are the details. Plasmids offered as cloning grade DNA As we begin testing out this service...with 210 plasmids from the collection. You can find the full list here. Most of these plasmids are empty...which plasmids you’d like to see as part of the service in the future using the Cloning Grade DNA Suggestion...blog posts from our Plasmids 101 blog series Learn about the different grades of DNA  Read about the various...

...from foreign DNA. The other component of these systems, methyltransferases, methylate DNA at particular... specific DNA sequences and companion endonucleases that recognizes and cleave the same DNA sequence if...stretches of DNA: Dam methylase adds a methyl group to the adenine of GATC stretches of DNA Dcm methylase...desired mutation into your plasmid of interest by PCR generates mutated plasmids with no methylation (there...Mutagenesis to Mutate Your Plasmids Resources on Addgene.org Check Out Our Plasmid Protocols Boost Your Molecular...Or considered why DpnI will degrade your template DNA from a PCR reaction but not the newly synthesized...the same--methylation! Read on to learn about how DNA methylation may affect your restriction digests. ...

...underrepresented plasmids (plasmids that were lost or “dropped out”) and overrepresented plasmids (plasmids that ...In addition to single plasmids, Addgene also distributes pooled plasmid libraries containing hundreds...transformed with the library DNA, they grow and replicate, and then their DNA is harvested, much like one...one would prep an individual plasmid. However, lots of library DNA is needed for screening experiments...so the scale of the DNA prep is larger. And since the library contains many plasmids, care must be taken...diversity. If the various plasmids in the library are differently sized, smaller plasmids may be amplified preferentially...pelleted for DNA extraction. Prepare library product for next-generation sequencing Once the DNA has been ...

...called DNA transposons. DNA transposons are commonly used as tools in the lab, so we’ll focus on DNA transposons...move antibiotic resistance genes between plasmids or from plasmids to the bacteria’s genome. In humans, if...Transposon: The transposon is the DNA sequence that moves around. This DNA sequence encodes the proteins ...BioRender.com.   Class II TEs: DNA transposons Class II TEs are also known as DNA transposons, since they do...break, and excises the transposon from the donor DNA. A DNA footprint is left behind. When the transposon-transposase...not leave behind a DNA footprint sequence after excision. piggyBac can mobilize DNA over 100 kB in size...transposase.  piggyBac plasmids    Tol2  Tol2 was the first reported active DNA transposon in vertebrates...

...Commercial DNA Ladder Penn State Ladder: Homemade Plasmid Prep Penn State Ladder: Commercial Plasmid Prep Kit...Two plasmids that can be used to make inexpensive 100 bp or 1 kb DNA molecular weight ladders were recently...developed the plasmids, pPSU1 and pPSU2. When restriction digested with PstI or EcoRV, these plasmids generate...Verifying Your Plasmid Table 1: Comparing Typical Comericial Ladders with the PSU Ladders DNA Lader Commercial...interested in making your own DNA ladder? You can find the Tan Lab’s DNA ladder prep protocol here and...Johnston, J. L., & Tan, S. (2017). The pPSU Plasmids for Generating DNA Molecular Weight Markers. Scientific ...generate 100 bp or 1 kb DNA ladders, respectively. Unlike many commercially available ladders, the 100 bp ladder...

... 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...