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We narrowed to 956 results for: REP

Showing: 321 - 340 of 956 results

  1. Plasmids 101: Dimers and Multimers

    Type
    Blog Post
    ...additional origins of replication compared to monomers, they are also able to replicate at a higher frequency...restriction sites in multimers usually repeat in tandem along with the repeated plasmid sequence, giving greater...much longer than expected, and the sequence was repeated two or more times in tandem. Such plasmids are...produces a multimeric form of the plasmid with repeating sequences. In some specific cases, multimerization...large inserts (>7 kb)(Berg et al., 1989), direct repeats (Ribeiro et al., 2009), and at high copy numbers...<300 bp) which cannot distinguish between large repeat sequences within a plasmid, it cannot be used to...sequencing. These longer reads are able to detect large repeat sequences within a plasmid and, therefore, allow...

  2. A Look at Addgene's QC Process

    Type
    Blog Post
    ...process ensures that any potential sequence discrepancies can be caught prior to distribution. Catching...issue? At Addgene, a QC issue is defined as a discrepancy that we believe may affect the function of the...decisions when analyzing the wide variety of discrepancies encountered in plasmids deposited to Addgene...resolve. Sequencing and assembly issues Sequence discrepancies are not always a result of mutations in the ... platform, such as GC-rich regions and highly repetitive sequences and/or features. Most of these regions...will contact the depositing lab to review the discrepancies. Often, the identified issues will not affect...requesting scientists aware of the identified discrepancy in the plasmid, and that it is not known to impact...

  3. Hot Plasmids February 2024

    Type
    Blog Post
    ...the new plasmids, antibodies, and viral preps in the repository through our Hot Plasmids articles.  Here's...PAGE editing with viral sgRNA targeting mCherry reporter (mChe), with co-incubation with assist peptide...variant (Piatkevich et al., 2023). Check out their preprint for more details. Not only will these mStayGold...doi.org/10.1038/s41592-023-02085-6. PMID: 38036853. Preprint: https://doi.org/10.21203/rs.3.rs-2941917/v1.  ...doi.org/10.1038/s41587-023-02018-w. PMID: 38081970. Preprint: https://doi.org/10.21203/rs.3.rs-2684100/v1.  ...proteins derived from StayGold. ResearchSquare (Preprint). https://doi.org/10.21203/rs.3.rs-3188559/v1..... CAP-Mac also robustly expressed fluorescent reporters in green monkeys and cultured human neurons, with...

  4. Plasmids for Endogenous Gene Tagging in Human Cells

    Type
    Blog Post
    ...sure that what we observe through the microscope represents reality as accurately as possible. This is especially...we have created an initial set of 10 plasmids representing 10 human genes and will add more to the collection...utilize the host cell’s inherent homology directed repair (HDR) process following the double strand break...and introducing the tag through homology directed repair To introduce the fluorescent tag to the cells, ...majority of the time the genome will attempt to repair itself without outside influence (non-homologous... the stem cell will seek out a template for the repair process. By flooding the cell with a plasmid containing...cell into using the introduced donor plasmid as a repair template. This results in the insertion of the ...

  5. Personal Protective Equipment (PPE) for BSL-1 and BSL-2 Labs

    Type
    Protocol
    ...been designed by the Addgene nonprofit plasmid repository and is being shared outside of Addgene for informational...informational purposes only. If you choose to reuse or repurpose this SOP in another location, please note that...

  6. Viral Vectors 101: Producing Your rAAV

    Type
    Blog Post
    ..., E4, and VA RNA genes; and the pRepCap plasmid containing replication factors and serotype-specific capsid...lentivirus, AAV does not possess the ability to replicate itself and depends on helper viruses like adenovirus...herpes simplex virus to facilitate efficient replication. The most common production system, however, ...been developed to determine the titers of rAAV preparations, such as dot blot, enzyme-linked immunosorbent...different titers are measured. A TCID50 measurement reports an infectious titer, meaning the concentration ...targeting the viral genome or inverted terminal repeats (ITR). You should always have a high-quality plasmid...overestimate of transducible units in the virus preparation.  rAAVs  Now your AAV virions are ready to store...

  7. Plasmids 101: Multicistronic Vectors

    Type
    Blog Post
    ...along with a reporter, such as a fluorescence gene or a resistance gene. These reporters allow you to ...multicistronic element(s), or you could replace one of the reporter genes above with your gene or genes of... using IRES elements. Further, scientists have reported lower expression of the downstream cistron due...eukaryotic cells, and, although some scientists report close to 100% cleavage with some of these peptides...that already has the multicistronic element and reporter cloned in. In these plasmids you would simply ... 2A element (depending on the placement of the reporter gene). Addgene's collection offers a variety of... are designed to have one or more of the genes replaced.  Additionally, 2A peptides could be PCR-cloned...

  8. Optogenetics + CRISPR, Using Light to Control Genome Editing

    Type
    Blog Post
    ...that can be repaired by either non-homologous end joining (NHEJ) or homology directed repair (HDR). This...photoactivatable repressor (dCas9). The activity of all variants was reversible and repeatable. Since these... spatiotemporal control, reversibility, and repeatability. The system developed by the Sata lab is composed...could be spatially controlled in a reversible and repeatable manner. With their light-activated CRISPR/Cas9...The system was also shown to be reversible and repeatable when blue light (450nm) was cycled on-off-on....system, Ye’s lab could edit a tdTomato fluorescent reporter gene in mice using Minicircle DNA vectors encoding...seen in the figure below. Next, by using a dual reporter fluorescence assay, Hemphill et al. demonstrated...

  9. 2023 and 2024: Reflections and Looking Ahead

    Type
    Blog Post
    ... countries, into our repository, while expanding global access to our repository through our distributor...At 3500 plasmids, this represents the largest single deposit in our repository and was a three–year project...14 deposits, that became available through our repository in 2023.  In June, we announced a new partnership... launch our Antibody Data Hub, an open-access repository for antibody validation data. We’ll continue ...expanding international access to the Addgene repository, partially through work with our distributors...we are now able to ship rabies plasmids in our repository to international requestors.    I would be remiss... used by our requestors, and supported by our repository. You can enjoy these stories, and other 20th ...

  10. What's the Best Way to Elute and Store Your Plasmid DNA?

    Type
    Blog Post
    ...sites if in double stranded DNA are repaired by base excision repair, but can lead to mutations in single...noticed that there are many options to elute your DNA prep. You might see protocols that recommend eluting ...single stranded DNA undergoing replication.   DNase on the other hand can digest and degrade the double ... buffer for preserving the stability of your preparation for a long time. Tris buffer controls the pH,... if the priority is to directly use your DNA preparation in any downstream application involving enzymatic...buffer is that it allows better control of the pH preparation stabilizing the plasmid, and still allowing most...at -20°C or even at -80°C, in this case your preparation can be eluted in water or in your buffer of preference...

  11. Getting the Most from Your Lentiviral Transduction

    Type
    Blog Post
    ...storage, viral preps should be divided into single-use aliquots, and stored at -80°C. Some reports suggest rapidly... time consuming, difficult, and lacking in reproducibility. Read on for some tips for getting the most...Obtain a vial of the desired cell line from a reputable distributor such as the American Type Culture ...you may want to consider concentrating your viral prep. Storage can also play a role in the success of ..., it is best to avoid freeze-thawing lentiviral preps unless absolutely necessary. Many labs try to use...this blog post for tips on titering your lentiviral prep!) and infect. While this approach is a great start...transducing with a range of dilutions of your lentiviral prep. Hopefully, by using more concentrated dilutions...

  12. Viral Genome Sequencing - A One Stop Shop for AAV Identity and Serotype Confirmation

    Type
    Blog Post
    ...confirm the identity and serotype of our AAV preparations. We recently published a description of the ... For labs that produce a small number of AAV preparations this level of quality control is sufficient ...facilities producing a large number of viral preparations in parallel, there is a very real risk of mixing...AAVs and the TA-based ligation and tagmentation preparation approaches described previously require a preliminary...effective substrates for tagmentation-based library preparation procedures. This finding eliminates the double-stranding...simplified approach, we sequenced over 100 AAV vector preparations and showed that VGS can quickly and reliably... script that can confirm the serotype of AAV preparations.  Using viral genome sequencing to determine...

  13. Celebrate Open Data Day with Addgene’s AAV Data Hub!

    Type
    Blog Post
    ...platform for scientists using Addgene AAV viral preps to easily find and share useful technical data. ...tools. This word cloud (using wordart.com) represents contributed data on brain injection site from...15 minutes or less if you have all of your data prepared. All of these questions relate to factors that...that you should have already recorded as good reproducible and record-keeping practice. In fact, when you...did you use? For animal subjects it is helpful to report the genetic background and catalog number or RRID...exactly which strain you used so if they wish to replicate your experiment, they know exactly where to get...take negative data, which is equally important to report. We recently published a paper in Frontiers in ...

  14. Hot Plasmids September 2018 - Optogenetics, RNA Localization, Fluorescent Protein, and Base-editing Tools

    Type
    Blog Post
    ...ACE) in real-time. The reporter constitutively expresses eGFP to indicate reporter abundance, and mCherry...highlight a subset of the new plasmids in the repository through our hot plasmids articles. These articles...and N177F mutations). Its excitation maximum is reported as 603 nm, while the emission maximum is 675 nm... ACE system: A versatile in vivo fluorescent reporter for rapid quantification of base-editing and Cas9...editing tools, the Reuben Harris lab has deposited a reporter plasmid that allows for rapid fluorescence-based... out a frame-shift mutation within the mCherry reporter. Quantification of editing efficiencies simply...containing rat APOBEC1. Finally, they show that the ACE reporter can also quantify Cas9 cleavage, thus exhibiting...

  15. Thoughts on the Future of Research 2015 Boston Symposium

    Type
    Blog Post
    ...can treat the underrepresented individuals too nicely. When the work of the underrepresented individual ... the academic research environment is setup to prepare trainees for further work in academia. In fact ...Increase the number of science masters programs to prepare scientists for positions outside of academia Require...(the current median postdoc starting salary as reported by Paula Stephan is $40,000) Increase Knowledge... Duchin provided a concrete example of how underrepresented individuals are discluded/not treated equitably...well-meaning mentors often fail to judge the work of underrepresented groups under their mentorship with the same...third party, it can come as a shock to the underrepresented individual. In an attempt to be nice, the ...

  16. 25 Work From Home Tips for Scientists

    Type
    Blog Post
    ...while meditating right before bed will help you prepare for sleep. At Addgene, our mindfulness group is...more about career paths and things you can do to prepare. Adapt to WFH while parenting Keep your kids...career exploration site Inventing Heron has a radio reporter challenge, and code.org has coding tutorials for... for all ages. Organize your data and prepare publications or your dissertation Organize data that you...write introductions to potential publications or prepare a review article. These are both options that can... your defense less stressful. Think about reproducibility best practices Support your future experiments...experiments and other researchers by implementing reproducibility best practices in your research. For a place...

  17. Using AAV for Neuronal Tracing

    Type
    Blog Post
    ... are replication incompetent and cannot readily be passed on to subsequent cells via replication like ...usually spread unidirectionally, there are some reports of bi-directionality. For predominately anterograde...cleft is usually severely impaired, and only few reports exist about dye molecules that are transported ...infection to the central nervous system where it replicates, spreads, causes neurotoxicity, and is lethal...HSV) also migrates through neurons where it can replicate and by doing so spread across several synaptic...cells are labeled overtime due to the continued replication of the virus. While this can be useful, if too...single-stranded DNA molecule and AAV vectors are replication deficient (i.e. they cannot easily spread between...

  18. Dos & Don'ts When Publishing a Scientific Manuscript

    Type
    Blog Post
    ...enough information to replicate your experiments, making research more reproducible and your results more...scientific communities. The ARRIVE guidelines for the reporting of laboratory animals during in vivo research ...research and the CARE guidelines for case reports are two sets of guidelines frequently used by editors. Providing...communities aim to significantly improve the reproducibility and reliability of science publications. Peer...s name and affiliations, and the entire referee report are permanently attached to the published article...and readers are able to comment on the referee reports. If the reviewers suggest that the manuscript requires...article, while the first version and the referee reports remain available online. Open-peer reviewing is...

  19. Antibodies 101: Multiplex Immunofluorescence

    Type
    Blog Post
    ...consider using a different antibody.  Prepare a positive control Prepare and label a sample that expresses... and secondary antibody labeling and imaging, repeating for each target. Alternatively, you can perform...paraformaldehyde-fixed cell samples, or other preparations. Sequential IF For a sequential IF experiment...wash, add secondary, image again, inactivate, and repeat these cycles for all your targets. Of course, you... ICC validated antibodies from Addgene), or be prepared to run some extra controls and validate the antibody...then label that with fluorescently-conjugated streptavidin to get more fluorophores per antibody, or conjugate...the offending red-green image, go to Image>Color>Replace Red with Magenta. Or you can use Color>Split Channels...

  20. Corralling and Cornering the Goldilocks Antibody

    Type
    Blog Post
    ...Unreliable antibodies cause research that cannot be reproduced and projects that cannot be completed. A 2015...data that they are a long-term solution to reproducibility and cost-effectiveness for broad and large ...criterion that includes accuracy, strength, and reproducibility. We test the antibodies on human and mouse ...comparison with two atlases enables us to see the reproducibility and accuracy while screening. We also compare...exposure time closer to the negative control. Reproducibility  In order to attain non-bias assessment, multiple...and exposure times. To test the antibody’s reproducibility, each antibody is independently screened twice...References and resources References Baker M. Reproducibility crisis: Blame it on the antibodies. Nature....
Showing: 321 - 340 of 956 results