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We narrowed to 79 results for: plasmid cloning by pcr

Showing: 21 - 30 of 79 results

  1. Easing the Protein Purification Process with pCri

    Type
    Blog Post
    Published
    June 19, 2015, 3:08 p.m.
    ... multiplexing. pCri plasmids are divided into two sets, each of which includes plasmids with different... As reviewed in our Plasmids 101 series, proteins are overexpressed from a plasmid construct, most often...C-terminal. Each set of plasmids uses the same restriction enzymes for cloning (see schematic below), ...on the Addgene Blog Plasmids 101: E. coli strains for protein expression Plasmids 101: Protein tags ... introduction to pCri, please see the original publication cited below. pCri plasmids are available either...on these and other tags used in the pCri system, please see Plasmids 101: Protein Tags. Beyond E. coli...Bacillus subtilis and Pichia pastoris pCri also includes plasmids designed for protein expression in systems...

  2. The time and cost required to make a plasmid

    Type
    Blog Post
    Published
    March 23, 2023, 1:15 p.m.
    ... long it takes to make a plasmid? Or how much time you have to spend cloning before you can start your...cost required to make a plasmid. In 2023 (and at closer to 2 million plasmids shared than not!), we think...great Plasmid Debate of ’18, Blue Flame depositor John Doench calculated the time to make a plasmid. Doench...simple insertion of a PCR product into a vector. The time it takes to make a plasmid could increase dramatically... make a plasmid? Doench also calculated the cost of consumable reagents used to make a plasmid, which ... to make plasmids that already exist, and we significantly reduce costs of producing plasmids with high-throughput...lot of plasmids, it’s almost always faster to order from us!) Ask the PI If you want a plasmid for free...

  3. Lambda Red: A Homologous Recombination-based Technique for Genetic Engineering

    Type
    Blog Post
    Published
    Dec. 15, 2016, 3:57 p.m.
    ...Restriction enzyme cloning is the workhorse of molecular cloning; however, one of its biggest limitations...sequence. This means cloning occurs in vivo, as compared to restriction enzyme cloning where the genetic ...background via P1 transduction. Plasmid Expressing lambda red genes from a plasmid allows for a mobile recombineering...recombinant clones are generated.  A simple way to do this is to express lambda red genes from a plasmid with...desired modification. Colony PCR can be used to screen for positive clones in most cases, and restriction... is an alternative method that can be used for cloning or genome engineering and is based on homologous... flexibility to modify the E. coli chromosome, plasmid DNA or BAC DNA.  To use the lambda red recombineering...

  4. CRISPR Protocol for Genomic Deletions in Mammalian Cell Lines [Video]

    Type
    Blog Post
    Published
    Feb. 18, 2015, 3:09 p.m.
    ...conventional PCR. Biallelically disrupted cells can be easily identified. Even in the case of clones with monoallelic...primer validation, and screening by PCR for biallelic deletion clones:    Like the video? The Orkin...polymorphism analysis, allele-specific PCR, T7EN1 cleavage assay, sequencing, RT-qPCR, or immunoblotting to detect...targeted deletions is to produce monoallelic deletion clones. Cells with just a single intact copy at a given...procedures. The video details how to transfect CRISPR plasmids into MEL cells by electroporation, sort your transfected...paper describes their methods for CRISPR design, cloning, and delivery, as well as their techniques for ... PubMed. More CRISPR Refereces: Browse CRISPR Plasmids CRISPR Information at Addgene More CRISPR-...

  5. Addgene's Top Blog Posts from 2020

    Type
    Blog Post
    Published
    Dec. 29, 2020, 2:15 p.m.
    ...and our new cloning grade DNA service. We've had 11 blog posts on COVID-19 featuring plasmid tools from...we also launched the cloning grade DNA service. This service features 210 plasmids: empty backbones, fluorescent...protein tags, SARS-CoV-2 plasmids, etc. It is our hope that by providing cloning grade DNA to you, we'd ...to Elute and Store Your Plasmid DNA? by Paolo Colombi Should I store my plasmid in TE, Tris, or water?...Digital PCR for AAV Quantitation by Kate Harten DeMaio We made the switch to droplet digital PCR to quantify...Here we cover five methods to do this. 5. Plasmids 101: Plasmid Incompatibility by Leah Schiesow If you're...save you days of time so that you can begin your cloning experiment right away. And without further ado,...

  6. PITChing MMEJ as an Alternative Route for Gene Editing

    Type
    Blog Post
    Published
    Feb. 23, 2016, 3:30 p.m.
    ...time spent cloning. In contrast, the short homology required by MMEJ can easily be added via PCR amplification...which facilitates easier cloning. An overview of PITCh. The PITCh plasmid contains a GFP-Puro cassette...cassette via PCR, and this construct is inserted into the PITCh vector via In-Fusion or SLIC cloning. Step 2...sequencing of puromycin resistant clones, they found that 80% and 50% of clones displayed proper insertion at...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...month and a half, without the need for complicated cloning of homology arms. MMEJ: An Introduction There...

  7. Evolution of Lab Techniques

    Type
    Blog Post
    Published
    June 21, 2016, 2:30 p.m.
    ...Restriction Cloning Get Tips on Analyzing DNA Sequencing Results Check out Plasmid Cloning by PCR Resources...replaced by real-time PCR to answer the same experimental questions. Real-time PCR detects and quantifies.... Southern blotting and quantitative real-time PCR Southern blotting, named for its inventor Edward ...applications range from confirming the results of cloning or knockout experiments to forensics and clinical...process, rather than just at the end as in standard PCR. This allows the researcher to compare different ...requires a large amount of high-quality DNA, real-time PCR has several advantages including easier automation... have a lower false positive rate than real-time PCR (4). Restriction mapping and modern sequencing Restriction...

  8. Educational Planarian Tools for Teaching Developmental Biology Techniques

    Type
    Blog Post
    Published
    May 24, 2018, 1:20 p.m.
    ... genes cloned into plasmids to investigate for each of these planarian species. These plasmids all have...), and then use PCR and molecular cloning to insert the cDNA into the desired plasmid backbone. The Sánchez...experiments in planarians with their recent plasmid deposit. These plasmids enable two main types of experiments...dsRNA-induced RNAi experiments. Notably, this plasmid harbors a multi-cloning site flanked by T7 promoters to allow...Students can produce DNA templates via PCR off of the same plasmid and use these to create riboprobes for...they can easily generate off of a circular plasmid via PCR. Typically, to make these constructs, developmental...their own molecular cloning and investigating genes not in this collection, they may clone DNA sequences for...

  9. Deep Mutational Scanning with One Pot Saturation Mutagenesis

    Type
    Blog Post
    Published
    Feb. 22, 2017, 3:30 p.m.
    ...Error-prone PCR: This method amplifies the wild-type template with a “sloppy” version of PCR that results...mutagenesis (SDM) PCR, but with a twist since it relies on the sequential nicking, degradation, and PCR-generation...a universal primer is used for this round of PCR. The PCR product is digested with DpnI to remove residual... 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...map of your plasmid. If the sites are in opposite directions, you just need to use a plasmid with BbvCI...

  10. When Fidelity Matters: A frank discussion about ligase fidelity

    Type
    Blog Post
    Published
    March 6, 2018, 1:49 p.m.
    ...Learn New Plasmid Cloning Techniques Pushing the Limits of DNA Assembly Plasmid Cloning by PCR Resources...vector-insert joining for recombinant plasmid construction (restriction cloning), adaptor ligation for next generation..., Francis. "The ligase chain reaction in a PCR world." PCR Methods Appl 1.1 (1991): 5-16. PubMed PMID:...interest are annealed to target DNA (genomic DNA or a PCR amplified fragment) and treated with a high fidelity... T4 DNA Ligase in applications such as routine cloning or DNA library preparation. However, when a method...Genetic disease detection and DNA amplification using cloned thermostable ligase." Proceedings of the National...PMC50775. 6. Barany, Francis, and David H. Gelfand. "Cloning, overexpression and nucleotide sequence of a thermostable...
Showing: 21 - 30 of 79 results