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We narrowed to 457 results for: nsf

Showing: 121 - 140 of 457 results

  1. Special Delivery: Fluorophore Targeting for FRET Studies

    Type
    Blog Post
    ...what's it good for? Fluorescence resonance energy transfer (FRET) is a biophysical interaction between two...first fluorophore (the donor) is excited, it can transfer energy directly via resonance to the second fluorophore...develop a successful FRET-based assay since energy transfer only occurs when donor and acceptor are close ...Å away from the donor doesn’t result in energy transfer and thus, your specific FRET signal is unaffected...experiments will define the maximum and minimum energy transfer values you can expect, thereby establishing the... Fessenden, James D. "Förster resonance energy transfer measurements of ryanodine receptor type 1 structure...PMC3647052. 11. Stryer, Lubert. "Fluorescence energy transfer as a spectroscopic ruler."Annual review of biochemistry...

  2. Tips for Titering Your Lentiviral Preps

    Type
    Blog Post
    ...they ensure that the correct transfer plasmid was used in the transfection; if preparing several different... your packaging cell line, prepped your DNA, transfected and harvested your lentivirus. Now it’s time ...assaying for expression of a gene carried on the transfer plasmid or quantifying the number of viral copies...utilizes the antibiotic resistance genes carried on transfer plasmids. Target cells are transduced with serial...multiplicity of infection (MOI) for predicting gene transfer events." Genetic vaccines and therapy 2.1 (2004...

  3. Simplify Cloning with in vivo Assembly

    Type
    Blog Post
    ...false positive colonies. Transformation - the linear fragments are transformed into E. coli and plasmid...assembly of a circular plasmid in vitro, before transforming it into E. coli for propagation. However, while...using a simple two-step, 2 hr protocol prior to transformation (Figure 1). As this approach requires no special... encode homology arms to allow immediate co-transformation with linearized vector backbone. DpnI digestion...failed so far. What is most important is the transformation efficiency of the strain: when asking the cell...

  4. Plasmids 101: Choosing an Antibiotic Resistance Gene

    Type
    Blog Post
    ...in a plasmid. During the propagation and/or transformation process, some bacteria will either never receive... is nifty because the recovery period after transformation (the dreaded 60 minutes shaking at 37 degrees... available, cost-effective, timesaving for transformations. Cons: Prone to satellite colony formation ..., interchangeable with Amp, timesaving for transformations. Con: Expensive.   Kanamycin Kanamycin, or...selection gene. However, it does require a slower transformation recovery period than amp or carb (60 minutes...confers mammalian resistance to G418. Con: Slow transformation   Spectinomycin/streptomycin Spectinomycin...

  5. Lentiviral Vector Uses and Overview

    Type
    Blog Post
    ...of three plasmids: the packaging, envelope, and transfer plasmids. The packaging plasmid carried a mutated...of cells the vector could infect. Finally, the transfer plasmid encoded the desired transgene flanked ...packaging and host genome integration. After co-transfection of these plasmids, 293T cells released transgene-containing...-competent virus via recombination between the transfer and packaging plasmids does exist. To further ...work with primary cells or other difficult-to-transfect cells. Lentiviral barcoding libraries have been... 18;127(7):839-48. PubMed PMID: 26758916. 7. Transfusion independence and HMGA2 activation after gene ...

  7. Lab to Office Culture Shock

    Type
    Blog Post
    ...opportunity to discover and evolve your transferable skills. Transferable skills are the non-technical skills...formulate hypotheses and conduct experiments. You transfer liquids between tubes, look through microscopes...production, I didn’t really start to recognize my transferable (or “soft”) skills until I was out of the lab...lab to think about these--get started with our Transferable Skills Guide.     Over the last decade, my... blog Visit our careers topic page Read our transferable skills guide Read blog posts about career readiness...

  8. CRISPR-mediated Plant Base Editors

    Type
    Blog Post
    ...72 hours post-transformation/transfection in a transient assay. For stable transformation, regenerated ...deaminases (ecTadA), fused it with dCas9, and transformed it into E. coli harboring the defective CamR ...genome edited plants via Agrobacterium-mediated transformation. We also constructed smaller vectors suitable...suitable for transient assays using protoplast transformation. Cytosine base editors for plant There are several...

  9. Hot Plasmids: Summer 2024

    Type
    Blog Post
    ...editor consisting of dCas9 fused to a DNA methyltransferase — that can drive lasting transcriptional silencing...Histone tail for Autoinhibition Release of Methyltransferase (CHARM), with several advantages over the ...  Rather than overexpressing an exogenous methyltransferase for epigenetic silencing, which can be toxic...tail to recruit and stimulate the endogenous methyltransferase. The strategy is generalizable to a variety...D3L recruits the endogenous, autoinhibited methyltransferase DNMT3A to the genomic location specified by...DNA-binding protein. The ADD domain of the methyltransferase binds to H3K4me0 on D3L, releasing the autoinhibition...

  10. Technique: Probe Phage Genomes for Host Binding Proteins

    Type
    Blog Post
    ...certain modifications to DNA bases.  Ligate and transform your resultant plasmid libraries into chemically...chemically competent E. coli and analyze transformants for appropriately sized inserts. We consider a library...culture, dilute and plate the complete library of transformants for single colonies (this usually requires 10...10-20 plates) (see box below). This initial transformation should be performed using a highly competent...extract plasmid DNA directly from the colonies. Transform the resultant plasmid DNA into E. coli BL21 (DE3... protein production), then dilute and plate transformants for isolated colonies (150-200 per plate) across...

  11. PiggyBac-ing Through the Genome Editing Field

    Type
    Blog Post
    ...we have found that transfecting with PBx mRNA can boost efficiency over transfecting with plasmids in some...from a licensed distributor) allow you to quickly transfer a DNA sequence from the transposon vector to one...traditional method of making stable cell lines is to transfect, select, and isolate clones. This procedure is...integration efficiencies that are accomplished with transfections and are therefore virus-free. This is achieved...fully intact, while, with a standard DNA plasmid transfection, a double-stranded break may occur in your cargo...

  12. Spurred by COVID-19, Addgene Bridges Plasmid Sharing Between Academia and Industry

    Type
    Blog Post
    ...worked closely with the legal partners and Tech Transfer Offices at universities to make COVID-19 research-related...United States, and many universities and Technology Transfer Offices were, at least, temporarily closing, these...scientists, we’ve worked with many scientists, tech transfer offices, research foundations, and funding initiatives...them available to industry, please reach out at techtransfer@addgene.org and we will initiate a conversation...conversation with you and your technology transfer office to discuss the possibilities for expanded sharing. ...

  13. Antibodies 101: Validation

    Type
    Blog Post
    ...Interest (GOI), and transfect it into your favorite cell line. In parallel, you transfect cells with another...express your protein of interest endogenously and transfect them with a plasmid encoding your protein of interest...expect to see evidence of antibody binding in the transfected cells but not in control cells. But (there is...results show a lovely, strong band from the cells transfected with your protein of interest and show just the... the faintest band from the cells transfected with the other plasmid. Your antibody works! Multiple (Independent...

  15. Understanding GPCR Signaling Using TRUPATH

    Type
    Blog Post
    ...Resonance Energy Transfer (BRET). BRET is similar to Förster Resonance Energy Transfer (FRET), in which...fluorophore. The difference for BRET is that the energy transfer is between a luminescent donor and a fluorescent...dissociation by bioluminescence resonance energy transfer 2, or BRET2. With binding of the drug, the alpha...transducers. To use the TRUPATH system, cells are transfected with plasmids expressing the GPCR of interest...

  16. Fluorescent Proteins 101: Luciferases

    Type
    Blog Post
    ...luminescent) worlds Forster Resonance Energy Transfer (FRET) is the transfer of energy from an excited donor fluorophore...Resonance Energy Transfer (BRET) works the same way, except that energy is transferred from a luminescence... fluorescent protein close enough, energy is transferred by BRET and the fluorescent protein emits light...

  17. Plasmids 101: Sequence and Ligation Independent Cloning (SLIC)

    Type
    Blog Post
    ...strong enough to hold a plasmid together through transformation, so the cloned vector must be ligated. But ...nicks easily repairable by the bacteria after transformation. LIC is a reliable cloning method, but it is...recombination intermediate to bacteria, so it can be transformed as-is into cells. The cells then get to work ...is long enough to hold it together, it can be transformed into bacteria, which will efficiently repair ... annealed. The recombination intermediate is transformed into bacteria, which repair the plasmid. Created...

  18. Simple CRISPR-based Epigenetic Editing: dCas9-directed DNA Demethylation

    Type
    Blog Post
    ...Tethering dCas9 to the histone acetyltransferase p300 or the DNA methyltransferase DNMT3A allows researchers...demethylation?  Dividing cells depend on DNA methyltransferase 1 (DNMT1) to copy methylation marks from parental... al (2018). Genome-wide tracking of dCas9-methyltransferase footprints. Nat Commun 9, 597. https://doi.org...promoters by targeted steric blockage of DNA methyltransferase with CRISPR/dCas9. Nat Commun 12, 5711. https...

  19. Nanoblades: Tiny CRISPR Ninjas for Genome Editing Difficult Cells

    Type
    Blog Post
    ... Options for CRISPR delivery include plasmid transfection, RNP electroporation, and viral transduction... non-replicative. Nanoblades are produced by transfecting the following four components into 293T cells...lower off-target effects when compared to DNA transfection of gRNA and Cas9 expression plasmids. These ...having lower off-target edits compared to DNA transfection.  Homology directed repair (HDR) with Nanoblades...can then fuse with the target cell membrane, and transfer their CRISPR cargo into the zygote. As a proof-of-principle...

  20. Quickest Way to Deposit Plasmids: The Deposit Spreadsheet

    Type
    Blog Post
    ...easy — our tech transfer team will communicate with your organization’s technology transfer office or other... Send plasmid information to Addgene Material Transfer Agreement (MTA) approval  Send plasmids to Addgene...require that you send your samples to Addgene pre-transformed in this strain. Hazardous If the plasmid...samples to Addgene and contact your technology transfer office about the MTA. Pro Tip! Download a new...
Showing: 121 - 140 of 457 results