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  1. Interview: Hodaka Fujii on enChIP, New CRISPR Tools, and More

    Type
    Blog Post
    ...enChIP Plasmids? All Hodaka Fujii lab plasmids Sign up to receive email alerts when new plasmids from ... shopping for a wide range of plasmids. The merit of depositing plasmids to Addgene has been spectacular... researchers all over the world have requested plasmids for enChIP through Addgene. Retroviral Expression...user-friendly and precise. We will deposit the plasmids of these systems to Addgene in the near future...Community Addgene: In 2012 your lab first deposited plasmids with Addgene. In 2014, you joined Addgene's Advisory...

  2. Fluorescent Proteins 101: Green Fluorescent Protein (GFP)

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    Blog Post
    ...GFP is a ~27 kDa protein consisting of 238 amino acids derived from the crystal jellyfish Aequorea victoria...from a maturation reaction of three specific amino acids at the center of the protein (Ser65, Tyr66, and .... Scientists can easily utilize GFP-containing plasmids as a means to many functional ends. We’ve listed...each protein or domain being tested. Find FRET plasmids here. Split EGFP: An alternative to FRET, split... marking/selection: Expression constructs like plasmids often include GFP as a marker to help identify...serve as an alternative to antibiotic selection. Plasmids of this type may have the GFP under the control...techniques in fluorescent microscopy. Read More: All Plasmids 101 Articles Addgene's Fluorescent Protein Guide...

  3. Tips and Tricks for Using Golden Gate Modular Cloning (MoClo)

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    Blog Post
    ...blog Plasmids 101: Modular Cloning Plasmids 101: Modular Cloning Applications and Kits Plasmids 101: ...insert’ refers to plasmids carrying each part, ‘vector’ refers to acceptor plasmids, and the ‘amount of...avoid cross-contamination, we recommend retrieving plasmids from your original Addgene kit plate as few times...you will also likely need to miniprep a lot of plasmids from your bacterial stocks beforehand. You can...efficient and precise, positive colonies may contain plasmids with undesired ligations, so always screen multiple...

  4. Overcoming the AAV Size Limitation for CRISPR Delivery

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    Blog Post
    ...amino acids in size, which includes SpCas9, and shortern orthologs approximately 1000 amino acids in size... Staphylococcus aureus Cas9 (SaCas9, 1053 amino acids) displayed cleavage activity in mammalian cells....small enough to package into AAV. At 984 amino acids in length, Cas9 from Campylobacter jejuni (CjCas9...Neisseria meningitidis Cas9 (NmeCas9) at 1,082 amino acids, can also be packaged into AAV. NmeCas9 has the ... proteins with AAV, at just 1,108 and 986 amino acids, respectively, the size of both is within AAV’s ...capabilities of full-length SpCas9. This set of plasmids includes AAV-Cas9C-VPR for targeted gene activation...Addgene.org Check out our CRISPR Guide Find CRISPR Plasmids ...

  5. Viral Vectors 101: Preparing Pooled Libraries

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    Blog Post
    ...anyone who has worked with individual plasmids knows, some plasmids amplify well and others don’t. The size...pooled library, which contains a collection of plasmids in a single tube, or a pre-made viral prep library... prepare the libraries for you.  Starting from plasmids requires amplification, verification, and lentiviral...library and appropriate packaging and envelope plasmids into HEK cells allows production of lentivirus...pooled library has hundreds to millions of distinct plasmids, all of which need to be represented in the same... verify via NGS now than to learn some of your plasmids didn’t amplify after your screen is completed!...need to infect many more cells than the number of plasmids in the library.  Spinfection If you have a large...

  6. What's New in CRISPR - September 2019

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    Blog Post
    ...Read the paper in Nucleic Acids Research Find the drug inducible Cas9 plasmids E. coli genome-wide inhibition...CRISPR guide Find CRISPR plasmids by function Find recently deposited plasmids ...series, we’ll highlight a few of the new CRISPR plasmids available at Addgene. We will still periodically...domain needed for DNA unwinding. They’ve deposited plasmids for active and inactive CasX for E. coli and human...expression. Read the paper in Nature Find the CasX plasmids Drug inducible transcriptional activation and...

  7. Running for Rare Disease, Running for FOP, Running for AJ

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    Blog Post
    ...CRIPSR Resources on Addgene.org Find ACVR1 Plasmids Find Plasmids for Disease Research on OurSpecial Collections...decided it was time to get in shape. After three kids, I had put on typical middle age weight and “exercise...Boston Marathon, he wanted to run and play with my kids like any other youngster. His parents, Kristi and...not be able to run around and play like the other kids his age. A potential FOP cure But, there is HOPE...two ways. First, Addgene is distributing ACVR1 Plasmids from a variety of research labs and hopes that...pathways that impinge upon FOP to deposit their plasmids with Addgene and join us in the fight against ...

  8. Viral Vectors 101: Parts of the AAV Transfer Plasmid

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    Blog Post
    ...experiments, making AAV starts with some plasmids. You just need three plasmids to start making AAV:  the packaging...plasmid. ITR sequences are 145 bases each and AAV plasmids have two ITR sequences. The DNA sequence between...specific (Haery et al., 2019). Most AAV transfer plasmids have a promoter, but this element isn’t required...transgene and are located between the ITRs. Transfer plasmids often have two pairs of lox sites which are part...forms a tertiary structure when transcribed which aids in nuclear export of mRNA and helps increase the...ITR.  polyA  The polyadenylation or polyA signal aids in the nuclear export of RNA and RNA translation...

  9. Pushing the Limits of DNA Assembly

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    Blog Post
    ...to Learn More about DNA Assembly? Check Out Our Plasmids 101 eBook! One of the features that makes NEBuilder...fragments (containing regions of homology) cut from plasmids via restriction enzyme digestion, and used directly...plasmid with the gene of interest (search Addgene plasmids by gene here), designing primers with your desired... desired edits to the target region. Assembled plasmids can be directly transformed into E.coli for plating...Other Long-Homology Based Cloning Methods Screen Plasmids by Colony PCR Addtional Resources on Addgene....Find Validated gRNAs Browse All Plasmids ... plasmids...

  10. Multiplex Genome Editing with CRISPR-Cpf1

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    Blog Post
    ...transfection of single plasmids vs pooled plasmids; for multiple edits, single plasmids are generally more...sites. This eliminates bulk from crRNA expression plasmids resulting in smaller, more streamlined vectors...versus 2.4% of cells transfected with a pool of plasmids that contained one crRNA per plasmid plus a Cpf1...your research? Check out the Cpf1 multiplexing plasmids available from Addgene. If you have any questions... Our CRISPR Multiplexing Resources Find CRISPR Plasmids for Your Research Catch up on Your CRISPR Background...

  11. Protein Tagging with CRISPR/Cas9: A Conversation with Mendenhall and Myers

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    Blog Post
    ...alerts when plasmids become available.  We’ve also had some requests for variations on the plasmids we’ve developed...and Myers for speaking with us! Find the Plasmids: Plasmids for protein tagging with CRISPR Additional... available at Addgene. For more details on the plasmids that are currently available along with detailed...results. The FLAG epitope is quite small—20 amino acids—so we don’t think it interferes with the function...Addgene: Why was it important to you to make these plasmids available to other scientists quickly? Myers: ...

  12. Kiran Musunuru on the Newest TALEN Genome-Editing System

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    Blog Post
    ...effector nuclease (TALEN) kit consisting of 834 plasmids. The researchers showed they could use their TALEN...particular DNA sequence? Musunuru: The kit includes 832 plasmids containing prefabricated pieces that allow you...the genome in rapid fashion. The two remaining plasmids in the kit have the rest of the protein in place...fluorescent proteins used to sort the cells after the plasmids have been introduced by transfection or electroporation...protein that binds those 15 base pairs is take four plasmids out of the library in the appropriate combination...testified by the fact that the TALEN kit includes 834 plasmids for the pieces you need to assemble. Our system...Cas9 (also available from Addgene) only has two plasmids. In many cases, CRISPR may be more efficient. ...

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

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    Blog Post
    ...residues in Thermus thermophilus DNA ligase." Nucleic acids research 24.15 (1996): 3079-3085. PubMed PMID: 8760897...of gaps in duplex DNA by T4 DNA ligase." Nucleic acids research 10.5 (1982): 1425-1437. PubMed PMID: 7041091...one-nucleotide gaps can be sealed by T4 DNA ligase." Nucleic acids research 15.21 (1987): 8755-8771. PubMed PMID: 3684572...detection of mononucleotide repeat sequences." Nucleic acids research 27.24 (1999): e40-e47. PubMed PMID: 10572192...comprehensive profiling of DNA ligase fidelity." Nucleic acids research 44.2 (2015): e14-e14. PubMed PMID: 26365241...by interrogating minor groove contacts." Nucleic acids research 32.15 (2004): 4503-4511. PubMed PMID: 15328364...fidelity of Thermus thermophilus DNA ligase." Nucleic acids research 24.15 (1996): 3071-3078. PubMed PMID: 8760896...

  14. Plasmid-based Recombinant Monoclonal Antibodies: What They Are and Why You Should Be Excited About Them

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    Blog Post
    ...Recombinant monoclonal antibodies are encoded in plasmids and produce a single antibody recognizing one ...then synthesize the genes that encode those amino acids (Tran et al., 2016). Each antibody would then be...laboratories and companies have already begun creating plasmids encoding affinity reagents. These include conventional...performed will be useful in perpetuity.  Second, plasmids are easy to store and share, making recombinant...limited in supply and producing antibodies from plasmids is much kinder to our animal friends! Finally,...genetically-encoded research tools, it is shocking that the plasmids and sequences for antibodies are not shared. For...useful variants of Cas9. By providing access to the plasmids encoding antibodies, scientists will be able to...

  15. Tips for a 1st time AAV user (by a Rookie AAV user)

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    Blog Post
    ... cells with AAV packaging plasmids. The cells need three different plasmids to produce AAV: 1) the RepCap...going to lie, AAV transfer plasmids are kind of a pain to work with. These plasmids contain ITRs which are...sites). Another way is to transform AAV transfer plasmids into bacterial strains, like NEB Stable. NEB Stable...Stable competent cells lack RecA, a protein that aids in the homologous recombination of duplicated regions...handling your AAV plasmid standard and AAV transfer plasmids. It’s easy to contaminate a workspace with plasmid...

  16. Fluorescent Tagging of Endogenous Genes with SapTrap

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    Blog Post
    ...tag and homology arms). Digestion of the donor plasmids with SapI allows the tag, selectable marker, and...to be released (Fig. 1, Step 2-3). Since donor plasmids of the same type will produce the same unique ...different combinations of connector and tag donor plasmids from the library can be used to generate functionally.... The CRISPaint toolkit includes several donor plasmids for protein tagging, including luciferase (NanoLuc...Cell Science recently developed a collection of plasmids to fluorescently tag markers of cellular structures...structures in mammalian cells. These plasmids use fluorescent proteins flanked by long regions of homology...protein tagging, please visit our website CRISPR/Cas Plasmids - Protein Tagging. Beyond simply monitoring protein...

  17. Sharing Your Lab Protocols: Using Apps to Save Time & Track Your Experiments

    Type
    Blog Post
    ...be found in our Plasmids 101 blog series. Want to learn more about sharing your plasmids via Addgene? Check... of the repository’s most frequently requested plasmids. I wondered how many scientists have used the ...http://www.addgene.org/ If you’re working with plasmids and you decide to share them using Addgene’s repository...biology educational resources, such as protocols, Plasmids 101 articles on various plasmid features, and ...scientists to upload protocols when they deposit their plasmids to the repository. These protocols show up on ...plasmid pages and can help scientists use these plasmids more successfully. And I’ll continue to add some...

  18. Selecting Your Plasmid Purification Kit

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    Blog Post
    ...throughput needed. Large constructs – Large plasmids, cosmids, or BACs, can be tricky to purify due to their...their normal kit protocols.  Yeast plasmids – If you’re extracting plasmids from yeast, instead of bacteria...purification protocols requires less plastic and avoids the use of more hazardous reagents like guanidine...environmental impact factor label Non-silica based, avoids use of chaotropic salts   What are all these...specifically optimized for purification of these large plasmids, while others have suggested modifications to ...Different Purity, Different Quantities, Different Uses Plasmids 101: Optimizing Plasmid Yields Bringing Sustainable...

  19. Introducing Addgene's Microbiology Pages

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    Blog Post
    ... To make it easier for you to find plasmids for your work, plasmids in the Microbiology Collection are...Microbiology Collection page, you can browse for plasmids by species and find our guides, plasmid kits and...resources for microbial research. Kits, guides, plasmids, and more We have Plasmid Kits for microbial work...us at [email protected]. If you are looking for plasmids used in E. coli and yeast, you’re in luck! Addgene...develop resources for your community! To contribute plasmids made in your lab, see www.addgene.org/deposit ...

  20. CRISPR Antimicrobials

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    Blog Post
    ... plasmid and phagemid delivery systems. Phagemids are plasmids containing a phage origin of replication...plasmid-based, CRISPR systems (Find the Citorik Plasmids here). In both cases, they used 1-2 CRISPR crRNAs...single base pair change in DNA gyrase, CRISPR phagemids were cytotoxic only for resistant bacteria. Bikard...replication-deficient. Thus, a very large number of phagemids would be needed for an in vivo therapy. Another...252409288. PubMed Central: PMCID PMC4˚237163. Find plasmids from this paper at Addgene. 2. Bikard, David,...Additional Resources on Addgene.org Find CRISPR Plasmids for Your Research Read Our CRISPR Guide Pages ...
Showing: 241 - 260 of 878 results