We narrowed to 498 results for: plasmid dna

... 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...

...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 ...

...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...

...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...

...deposited plasmids. To resolve QC issues, assembly issues, and difficult-to-sequence plasmids, other sequencing...ddNTP) so that after its addition, DNA synthesis stops. The resulting DNA fragments are pooled and then separated...it to the plasmid information or depositor-provided sequences to confirm the correct plasmid components...Addgene The Power Behind NGS Plasmid Validation: seqWell Addgene’s Tips for Plasmid Quality Control Additional...general overview of the QC process at Addgene. All plasmids go through this same initial QC process using ...in 1977 by Fred Sanger, was the first widely used DNA sequencing technology. The process relies on “sequencing-by-synthesis... where each base is recognized and recorded as a DNA strand is synthesized (Figure 1). Sanger sequencing...

...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...

...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...

...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 ...

...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...

...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...

...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 ...

...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...

...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...