Skip to main content
Addgene
Open Menu

Enter a sequence to perform a BLAST-based search of Addgene plasmid sequence databases.

Information

In Category

We narrowed to 1,064 results for: tra

Showing: 321 - 340 of 1064 results

  1. An “elegans” Approach to Better CRISPR/Cas9 Editing Efficiency

    Type
    Blog Post
    ...CRISPR/Cas9 editing strategies in C. elegans As brilliantly and irreverently illustrated in the parable of...development of toolkits such as the SapTrap CRISPR/Cas Toolkit and SapTrap-SEC kit for C. elegans genome engineering...encode the gRNA transcript and the repair template of your choosing. Tools such as SapTrap Builder greatly...Genome Editing with Short Single-Stranded and Long, Partially Single-Stranded DNA Donors in Caenorhabditis...Emerging CRISPR/Cas9 editing technologies have transformed the palette of experiments possible in a wide...experimental manipulation and screening.  A co-selection strategy for CRISPR editing in C. elegans improves efficiency...Jordan Ward, UCSF. In 2015 (Ward, 2015), I demonstrated that selection for repair of a temperature-sensitive...

  2. IDP and your PI: A Roadmap for Career Planning and Personal Development

    Type
    Blog Post
    ...blog posts: Extracurricular Activities for a Strong Science Career Path 7 Simple Strategies to Resolve ...Good Manager Anyway Developing Transferable Skills During Science Training ...key paper, or figuring out a career path? Many trainees are developing Individual Development Plans (IDP's...then formulate a plan to further develop your transferable skills and reach your career goals.      Although...used in isolation. Without clear dialogue between trainee and PI, it’s easy to miss out on beneficial opportunities...DePace holds yearly planning meetings with each trainee to create a supportive, growth-oriented lab culture... each of which is filled out separately by the trainee and PI before the meeting. These worksheets, summarized...

  3. What’s in Your Tube?: A Quick Guide to Measuring DNA by Spectrophotometry

    Type
    Blog Post
    ...accurate measure of DNA concentration. This is especially problematic in low-concentration samples (samples ...pure your sample really is?  To assess the concentration and purity of nucleic acids, such as plasmids...spectrophotometers make it quick and easy to measure sample concentration and purity using only a tiny bit of the sample...— briefly Spectrophotometers measure how light travels through a sample — in this case, the absorbance...Nucleic acids exhibit a characteristic absorption spectra across this range of wavelengths with maximal absorption...instrument can calculate a sample’s nucleic acid concentration based on the sample’s absorption at 260 nm (...). Figure 1: Characteristic absorption spectra for pure DNA and likely contaminants found in DNA...

  4. dTAG - You're it!

    Type
    Blog Post
    ...Monitor neo-substrates Small molecule degraders may recruit CRBN to nonstandard or “neo-substrates.” We advise...al., Huang et al., Weintraub et al.), washout experiments (Nabet et al. and Weintraub et al.), chemical...et al.), transcriptional and epigenomic profiling studies (Nabet et al., Erb et al., Weintraub et al.),... et al., Huang et al., Weintraub et al.), knock-in (Nabet et al. and Weintraub et al.), and in vivo studies...degradation of endogenous proteins is an exciting strategy, it is difficult to apply this technique to oncoproteins...limitation, we created a generalizable tag-based strategy, which we refer to as the degradation tag (dTAG...targets in time scales that are not possible with traditional genetic approaches. In addition, the dTAG system...

  5. Plasmids 101: Shuttle Vectors

    Type
    Blog Post
    ...mammalian analogue of bacterial transformation is transfection. In stable transfection, DNA from the plasmid is...vectors delivered by transformation and transfection and left the business of transduction to our Viral Vectors... Plasmids 101: Transformation, Transduction, Bacterial Conjugation, and Transfection Plasmids 101: Choosing...to retain the vector as cells divide. In transient transfection, the vector remains episomal, meaning it...vector replication at all. In these cases, transient transfection may be sufficient for your needs even without...called transfer vectors. The gene of interest is cloned into a shuttle vector for transfection into producer...has seen before, showing how DNA sequences are translated into amino acids. And I was astonished that the...

  6. Hot Plasmids - November 2023

    Type
    Blog Post
    ... cargo Optogenetic proximity labeling and TransitID Tracking cellular history with protein ticker tape...also incorporated LOV-Turbo into TransitID experiments.  Find TransitID plasmids here! These two developments...Dynamic mapping of proteome trafficking within and between living cells by TransitID. Cell, 186(15), 3307–3324...in vitro and in vivo. Incorporating reverse transcriptase (RT) variants, PE6a and PE6b are 500–800 bp ...37612276.   Optogenetic proximity labeling and TransitID By Mike Lacy Proximity labeling can identify protein...naturally present in experimental systems. They demonstrated LOV-Turbo in neurons, in pulse-chase experiments...plasmids here! Next, Qin and Cheah et al. described TransitID, where two proximity labeling enzymes, TurboID...

  7. Viral Production at Addgene

    Type
    Blog Post
    ...of Pennsylvania Vector Core. Transfections are performed using the transfer plasmid, a plasmid encoding...are purified by iodixanol gradient ultracentrifugation and concentrated to purity and titers adequate for...ddPCR) titration protocol (modified from Wang et al., 2018). Briefly, 293T cells are transduced with serial...PCR with primers targeting the transfer plasmid used in the transfection. PCR products are analyzed by ...cell cultures are inspected for sterility. Transducibility Some viral vectors are tested in vitro and ...images).     Figure 2: AAV Pro cells were transduced with either pAAV-Ef1a-mCherry-IRES-Cre (55634...cleared by low-speed centrifugation and then by filtration through a 0.45 µm polyethersulfone (PES) membrane...

  8. pCXLE toolkit: Efficient episomal plasmid-based method to reprogram peripheral blood cells to iPSCs

    Type
    Blog Post
    ...hiPSCs for clinical purposes: mRNA transfection- this allows transient expression of the reprogramming factors...cells, thus expressing additional EBNA-1 transiently. The transiently expressed EBNA-1 increases protein expression...is easily and routinely drawn from patients in contrast to the process of collecting fibroblasts, which...insertional mutagenesis, which is the integration of transgenes into the genome that can cause disruption of ...non-integrating RNA virus Episomal vectors- extrachromosomally maintained DNA plasmids that express reprogramming...reprogramming factors To date, the mRNA transfection method has not been used successfully to derive iPSCs...encoding reprogramming factors behave as separate extrachromosomal elements in the nucleus of the target cell...

  9. Bacterial Toxin-antitoxin Systems as Molecular Biology Tools

    Type
    Blog Post
    ...required for hok translation. Here, Mok and Hok translation is coupled. Translation from the Shine-Dalgarno...more stable than the methyltransferase. When a plasmid is lost, the methyltransferase is degraded and/or...Science translational medicine 7.289 (2015): 289ra84-289ra84. PubMed PMID: 26019220. PubMed Central PMCID... by binding the GyrA subunit of DNA gyrase and trapping it in the cleaved complex. This results in DNA...ccdB gene die off and a higher percentage of transformants contain the ccdB-less construct. Other toxin-antitoxin...introduce multiple genetic modifications in the same strain. MazF expression is under control of the AOX1 promoter...composed of restriction endonucleases and methyltransferases. Restriction endonucleases cleave DNA at ...

  10. RUBY-Red Siliques

    Type
    Blog Post
    ...Selectable transgene markers for plant transformation fall into two main categories: antibiotic/herbicide...presence of a transgene instead, which can’t be done if your selection method kills the transgene free individual...Arabidopsis seed following floral dip transformation next to an untransformed seed from the same plant beginning...resistance genes usually involves killing the untransformed seedlings or callus (undifferentiated plant ...For example, with CRISPR-Cas9 gene editing, transformation may be identified by resistance to an antibiotic...antibiotic. Successful editing of transformants then typically occurs in T0 plants (from callus) or T1 plants...contaminate it. I have used it for selecting transformation events and am currently planning to use it ...

  11. Antibodies 101: ELISA (Enzyme-linked Immunosorbent Assay)

    Type
    Blog Post
    ... the reaction of HRP with its substrate, 3,3′,5,5′-Tetramethylbenzidine (TMB). The addition of hydrochloric...coating the bottom of the well), and then its concentration is detected through visualization of an enzymatic... molecule, such as biotin. Next, a conjugated substrate is added to the wells. In the case where the detection...enzyme horseradish peroxidase (HRP). Finally, the substrate of HRP is added to the wells. This reaction produces...produces a colorimetric reaction that reflects the concentration of your target protein in each well. The reaction...Sandwich ELISA to quantitatively measure protein concentration or antibody specificity via a colorimetric reaction...Competitive ELISA to quantitatively measure protein concentration or antibody specificity via a colorimetric reaction...

  12. The MTA at Addgene

    Type
    Blog Post
    ...the material transfer agreement, read on!  What is the MTA?  An MTA is simply a contract or agreement...Biological Material Transfer Agreement (UBMTA) is one of the most commonly used ones for transfers between academic...shine.  These types of agreements allow for centralized tracking when biological reagents are shared — both...for more information. Happy transferring!    Many thanks to our Tech Transfer team members Maura Coughlin...Addgene, then you’ve been involved in the material transfer agreement (MTA) process. Even though this process...the creation of a standardized agreement on the transfer of biological materials in 1995: the UBMTA. Because...most academic and nonprofit institutes, the Tech Transfer department is responsible for managing research...

  13. R Bodies: Membrane-Rupturing Microscopic Tools

    Type
    Blog Post
    ...endocytosis, vesicle trafficking, or muscle contractions) are produced by orchestrated movements of networks...explore their ability to penetrate other barriers (like vasculature or extracellular matrices) in response...PMID: 3759909. PubMed Central PMCID: PMC213417. 2. Schrallhammer, M. et al. Tracing the role of R-bodies...learn a lot from Nature’s simpler solutions to transmitting forces over long distances. For instance, how...machines R bodies can repeatedly extend and contract in response to changing pH, and they can rupture...

  14. Hot Plasmids: Summer 2025

    Type
    Blog Post
    ...neuronal tracing, anterograde tracing poses challenges, from non-selective anterograde transport to a lack...called Simultaneous Tracking of Recombinase Activation and Clonal Kinetics (STRACK) to study stem cell...well-suited for lung cells via airway administration. AAV11 can transduce neurons for retrograde labeling, ...ATLAS: a rationally designed anterograde transsynaptic tracer. Nature Methods, 22(5), 1101–1111. https...With this proof-of-concept, they believe that the strategy will be applicable with other TnpBs and in other...Viral delivery of an RNA-guided genome editor for transgene-free germline editing in Arabidopsis. Nature Plants...While many barcoding libraries are available for tracking cell lineage, different applications require careful...

  15. SARS-CoV-2/COVID-19 Detection Methods Based on CRISPR/Cas

    Type
    Blog Post
    ...and to do so, the WHO recommends one strategy: testing, tracking, and social distancing. With countries...are designed, each binding to one strand of the reverse-transcribed target sequence that are present close... the samples. First, RNA is extracted and subject to reverse transcription. This is then amplified by ...PCR using Taq Polymerase and then transcribed to provide RNA substrates for Cas13. Cas13, upon finding complementarity...block and 62°C heat block Approach: Viral RNA is extracted and subject to isothermal amplification by primers...cleaves the supplied fluorescently labelled ssDNA substrates. Upon cleavage, the fluorescence is no longer...directly using saliva without having to perform RNA extraction but that may work only if there is a high enough...

  16. Harnessing Bacterial Toxins for Allelic Exchange

    Type
    Blog Post
    ...rhamnose-inducible promoter, though Khetrapal et al. also demonstrated that the arabinose promoter worked...strong transcriptional terminator BBa_B1002 (from IGEM) upstream of the MCS to reduce transcriptional readthrough...Channarasappa, and Howard M. Salis. "Translation rate is controlled by coupled trade-offs between site accessibility.... PubMed PMID: 23360965. PubMed Central PMCID: PMC3748948.  Khetrapal, Varnica, et al. "A set of powerful...rely on exogenous enzymes, require multiple transformation and plasmid curing steps, and can generate ...toxins to facilitate efficient recombineering (Khetrapal et al., 2015). They created vectors for Lambda...tight (Giacalone et al., 2006). The Chen lab demonstrated excellent counter-selection which enabled very...

  17. Thoughts on the Future of Research 2015 Boston Symposium

    Type
    Blog Post
    ...benefits commensurate with work training and experience, better align trainee expectations with job outcomes...that fact that training in the academic research environment is setup to prepare trainees for further work...non-academic careers, much of the training they receive as postdocs is not transferrable to other environments. ... of trainees at low wages will still be there without restructuring. Positive Points for Trainees - Taking...information - all traits you are likely to find in graduate students and postdocs. Trainees however, need ... the mentor and this training is clearly not equitable to that of other trainees despite the fact that...temporary and defined period of mentored advanced training to enhance the professional skills and research...

  18. 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...membrane and deliver the substrate; however, secreted luciferase or alternative substrates can permit measurements...incompatible substrates can also allow multiplexing with experiments that supply both substrates and measure...production of light by a chemical reaction. In contrast, fluorescence occurs when a molecule is excited... Bioluminescent reporters require a chemical substrate (and sometimes cofactors) to be provided and are... they’ll keep emitting light as long as their substrate is available. Table 1 compares some general advantages...

  19. Viral Vectors 101: Viral Applications

    Type
    Blog Post
    ...Viral delivery is attractive due to the ease of transduction in difficult-to-transfect cell types ranging...higher than traditional transfection and random genomic integration of a plasmid. Viral transduction is also...overexpression of all human transcription factors, barcoded libraries to trace cell lineage, and so much...delivered with viral vectors instead of a traditional transfection method. This is because viral systems ...integrated into the genome of the target cell or transiently expressed. It’s important to note that for all...vector can express your construct in target cells transiently or constitutively depending on the virus used...also generally less toxic to cells than transfections, preserving cell viability. Library delivery Screening...

  20. Droplet Digital PCR for AAV Quantitation

    Type
    Blog Post
    ...droplets is used to calculate the concentration of the sample. This concentration can then be used to calculate...possible to measure the concentration of multiple targets simultaneously. For AAV titration, you should only ...tool for quantifying AAV for some time now. When titrating AAV by qPCR, viral DNA is amplified and monitored...For these reasons, we are in the process of transitioning our titering method from qPCR to ddPCR. Droplet...needed for your PCR experiment. The process to titrate AAV by ddPCR begins with diluting the virus. It...million.  After making the dilutions, they are transferred to a new plate containing the mastermix which...place.  When the PCR is complete, a droplet reader extracts the droplets from the plate and measures the fluorescence...
Showing: 321 - 340 of 1064 results