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We narrowed to 79 results for: plasmid cloning by pcr

Showing: 51 - 60 of 79 results

  1. Technique: Probe Phage Genomes for Host Binding Proteins

    Type
    Blog Post
    Published
    Sept. 29, 2016, 2:30 p.m.
    ... following colony PCR of 10-20 colonies using primers bordering the multiple cloning site of the vector...pool and extract plasmid DNA directly from the colonies. Transform the resultant plasmid DNA into E. coli...Visit Our Microbiology Pages Use the FX Cloning Sytem to Clone Your Gene of Interest Into a Variety of...binding proteins are hard to find Sequencing and cloning phage DNA can be difficult, in part because phages...gene-sized) fragments so that these fragments can be cloned into E. coli and subsequent proteins screened for...RBP gene without causing issues associated with cloning large fragments. After fragmentation, repair sheared...DNA bases.  Ligate and transform your resultant plasmid libraries into chemically competent E. coli and...

  2. 28 Hot Plasmid Technologies from 2015

    Type
    Blog Post
    Published
    Dec. 23, 2015, 3:30 p.m.
    ...standard entry clone. ORFs from an ORFeome Gateway library or any PCR-amplified ORF cloned into pDONR221...Multisite Gateway cloning. A desired insert is cloned using traditional restriction enzyme cloning to create ...amazing plasmid technologies developed by our community of depositors. With over 40,000 plasmids avaliable...amazing new plasmid tools that have come through our doors in 2015. Do you have a favorite new plasmid tool ...system plasmids are available through Addgene and are already very popular. Use these plasmids to increase...100% cloning efficiency and gene expression. The pUCXKT vector ensures that all screened plasmids contain... promoter-less Gateway® entry clones to be used with other entry clones encoding the ORFs of interest:...

  3. Plasmids 101: Optimizing Plasmid Yields

    Type
    Blog Post
    Published
    May 26, 2016, 2:30 p.m.
    ...more cells = more plasmid. Plasmids can vary in copy number, the number of plasmid copies supported within...have DNA left over for other applications, like PCR, cloning, transfection, or long-term storage. But what...where your plasmid yield is sub-optimal? If you are consistently getting sub-optimal plasmid yields from... optimize your plasmid prep We'll discuss the following techniques to increase plasmid yield. Keep in ...genotype, as these mutations improve plasmid stability and yield. If a plasmid is prone to recombination Use ... a strain optimized for plasmids with repeat elements, such as viral plasmids with inverted terminal repeats...co-purify with your plasmid DNA and lead to degradation of your prep. Many plasmid prep kits recommend...

  4. Production of Virus in Insect Versus Mammalian Cells

    Type
    Blog Post
    Published
    Nov. 5, 2024, 8:32 p.m.
    ...three plasmids — a transfer plasmid containing your gene of interest plus two packaging plasmids (three...only have to worry about cloning your transfer plasmid. The transfer plasmids have different packaging...three packaging plasmids for 3rd generation lentivirus production). The packaging plasmids are widely available...but they require cloning specific AAV or lentiviral machinery into a baculovirus plasmid. Production of ...of baculovirus requires only two plasmids — your transfer plasmid and the baculovirus construct, called...lentiviral packaging plasmids, bacmids are readily available, so the amount of cloning required is about ...titrated following purification using qPCR or digital droplet PCR. Insect cells Insect cells are a less...

  5. PiggyBac-ing Through the Genome Editing Field

    Type
    Blog Post
    Published
    May 31, 2016, 3:30 p.m.
    ...the ability to distribute plasmids with the piggyBac™ transposon. These plasmids, when combined with a source...useful resource. While Addgene cannot distribute plasmids with the piggyBac™ transposase itself, please ...cell lines is to transfect, select, and isolate clones. This procedure is cumbersome and inefficient, ...genome fully intact, while, with a standard DNA plasmid transfection, a double-stranded break may occur...differentiation process. Moreover, a splinkerette-PCR assay may be used to map the specific genetic loci...by utilizing a piggyBac™ transposon in a donor plasmid carrying the DNA edit of interest, along with a...piggyBac™ transposase enzyme [4]. By using a donor plasmid containing a negative selection marker such as ...

  6. Hassle-free 96-well Format Epitope Tagging Using Cas9 Ribonucleoprotein

    Type
    Blog Post
    Published
    June 28, 2018, 3:01 p.m.
    ...on the Addgene Blog Plasmids for endogenous gene tagging in human cells Plasmids 101: Protein Tags Fluorescent...from many pitfalls: they require construction of plasmid vectors and/or rely on cumbersome in vitro synthesis...7 days. The crRNA and tracrRNA are annealed on a PCR block and the annealed cr:tracrRNA is then mixed ...directly purchased and there is no need to construct plasmid vectors or synthesize/purify IVT sgRNA in the lab...using the csRNP method is as simple as doing routine PCR assays. Here is a simple to-do-list to follow when...Assemble csRNP: Anneal crRNA and tracrRNA using a PCR block. Add Cas9 protein to the annealed cr:tracrRNA...well plates: Characterize clonal lines by ICC and PCR genotyping (Sanger sequencing); use primers flanking...

  7. Deep Dive: qPCR

    Type
    Blog Post
    Published
    Sept. 29, 2022, 1:15 p.m.
    ...Addgene blog Digital Droplet PCR for AAV QuantificationPlasmid Cloning by PCR... Laperriere G;  Germain H. Droplet Digital PCR versus qPCR for gene expression analysis with low abundant...just getting started, check out our Introduction to PCR.) But if you're ready to go beyond the 101 and take...single molecule often work better for rapid-cycle PCR, such as the LightCycler, as it reduces the primer...concentrations and a temperature range) to predict PCR melting curves and dynamic melting profiles (Dwight...target genes can have different efficacies DART-PCR No Raw Data Statistically defined log-linear ...Biassoni R; Raso A. (eds.), Quantitative Real-Time PCR: Methods and Protocols, Methods in Molecular Biology...

  8. Tips for arabidopsis transformation

    Type
    Blog Post
    Published
    Oct. 25, 2018, 1:23 p.m.
    ...general steps for this method include: Cloning and transforming a plasmid into the bacterium Agrobacterium ....org Find plant plasmids Visit our plant resource page Deposit arabidopsis plasmids with Addgene ...available (Weigel and Glazebrook, 2002). Find plant plasmid resources at Addgene  Choosing an expression system...options, but here are a couple of my favorites. Plasmids from the Tsuyoshi Nakagawa lab are binary gateway...prefer Golden Gate assembly, there are a series of plasmids from the Dinneny lab that additionally allow for... Weigel and Glazebrook, 2002. Tips: Do a colony PCR on your agrobacterium: Getting a transformant takes...culture you are growing definitely contains your plasmid. Grow lots of Agrobacterium: The more agrobacterium...

  9. Selecting Your Plasmid Purification Kit

    Type
    Blog Post
    Published
    May 27, 2025, 1:15 p.m.
    ...your plasmid; separate plasmid DNA from unwanted cellular “debris”; and then clean-up the plasmid DNA ...debris from the plasmid, and then clean-up the plasmid so that you are left with pure plasmid DNA. Image created... help you choose the plasmid prep kit that is right for you. What type of plasmid prep do you need? As...As we covered in a previous post on plasmid preps, not all plasmid preps are the same. Your downstream...get transformants if the plasmid you are using is completely degraded. Cloning Grade DNA (high quality/...arise during the plasmid purification process. One is the possibility of damaging the plasmid DNA itself, ...carrying over contaminants from the plasmid prep process into the final plasmid eluate. Possible contaminants...

  10. 27 Hot Plasmids from 2016

    Type
    Blog Post
    Published
    Dec. 22, 2016, 3:03 p.m.
    ...eliminating the need for PCR or additional molecular cloning steps during plasmid assembly. This toolkit...Neurosci 2016. PubMed PMID: 26950006 Plasmid ID Plasmid Name Plasmid Type 74309 pcDNA3.0-TRPV4-p2A-ferritin-p2A-mCherry...highlight a subset of the new plasmids in the repository through our hot plasmids articles. These brief articles...features and applications of a partiular plasmid or set of plasmids. We hope that these articles make it ... you to find and use the plasmids you need. You can find all the hot plasmids from 2016 below. With over...over 45,000 plasmids, we can't write posts for every great plasmid that comes into the repository, but...write about your plasmids in a future blog post. No time to read? Listen to our hot plasmids segment on the...
Showing: 51 - 60 of 79 results