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

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We narrowed to 562 results for: PAC

Showing: 41 - 60 of 562 results

  1. Water Bath Protocol

    Type
    Protocol
    ...bath, and be sure to know what the maximum fill capacity is. Look for a “fill” line inside the water bath...

  2. Video Library

    Type
    Protocol
    ...personal protective equipment, setting up a clean workspace, and maintaining sterility while working. AAV ...

  3. CRISPR History and Development for Genome Engineering

    Type
    Collection
    ...contains many unique protospacer sequences that have homology to foreign DNA. Protospacers are separated by...Adaptive Immune System CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) sequences were initially...contain a species-specific sequence known as a protospacer adjacent motif (PAM). The CRISPR complex binds...sequence is only valid if it contains a special Protospacer Adjacent Motif (PAM) directly after where the... development has occurred at a truly astonishing pace, with work directed towards increasing on-target...fidelity Cas9 variants , SpCas9-HF, eSpCas9, and HypaCas9 display very low off-target activity due to rationally...SaCas9) is smaller than SpCas9 and more easily packaged in AAV. Cas9 orthologs also have distinct PAM ...

  4. Experimenting in an Open Source lab: from CRISPR to Cats

    Type
    Blog Post
    ...1,000 active makerspaces around the world. Hackerspaces.org maintains a list of active spaces near you. ...@derekja  Resources Hackerspace Website https://wiki.hackerspaces.org/Hackerspaces OpenWorm Project Website...manifested itself in the creation of Hackerspaces and Makerspaces back in 2007, which function as centres...with the OpenWorm Project One such space is the Victoria Makerspace in Canada, which I belong to. The ...  Funding Makerspaces One of the main challenges when starting a new project at a makerspace is finding... can find a makerspace somewhere near you. The place to start looking for your local space is the Hackerspace...competition. In 2010, he founded makerspace.ca, Victoria’s first hackerspace which now offers members and ...

  5. Genomic Deletions in Mammalian Cell Lines

    Type
    Collection
    ...consist of a 20-mer (“protospacer sequence”) upstream of an “NGG” sequence (“protospacer adjacent motif” or...Abstract The prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated...Figure 2A ). NOTE: In this example, sgRNA-A’s protospacer sequence and PAM happen to fall on the bottom...bottom (Crick) strand while sgRNA-B’s protospacer sequence and PAM fall on the top (Watson) strand ( Figure...will occur independent of orientation of the protospacer sequence/PAM relative to the top or bottom strand...However, if the first position of the 20-mer (protospacer sequence) is G, do not add another G ( e.g., ...cleavage site to ensure detection would not be impacted by a small indel at the sgRNA target site. Design...

  6. Supporting Reproducibility with a Connected ELN

    Type
    Blog Post
    ... take a look at the RSpace ELN.  The connected approach in action: how the RSpace ELN supports reproducibility...repositories. RSpace has been integrated with two of the five: Figshare and Dataverse, as well as the DSpace repository... reproduce an experiment documented in RSpace using the RSpace datasets deposited in Figshare or Dataverse...Rory is founder and CEO of Research Space, which provides the RSpace electronic lab notebook. Rory is based...contributed by Rory Macneil, founder of Research Space. There are many types of electronic lab notebooks...reproducibility RSpace was designed in response to requirements that emerged from intensive engagement with...byproduct of connectivity. Between 2012 and 2017 RSpace was developed with these principles in mind, and...

  7. Overcoming the Challenges of Lentiviral Production

    Type
    Blog Post
    ...generation packaging systems use 3 plasmids; an envelope plasmid that usually encodes VSV-G, a packaging plasmid...lentiviral packaging systems please see Addgene’s Lentiviral Guide. While the third generation packaging systems...requirement for additional DNA can impact the transfection efficiency of the packaging cell line and thereby affect...are transfected into a packaging cell line, usually a derivative of 293T. Packaging cells are incubated ...delivering genes via lentivirus is deciding what packaging system is best for a particular experiment. The...The newer generation lentiviral packaging systems were designed to prevent the formation of replication...reduce the risk of such events, newer lentiviral packaging systems divide the viral elements among multiple...

  8. Your Lentiviral Plasmid FAQs Answered

    Type
    Blog Post
    ...lentiviruses the same thing? Can I package lentivirus with retroviral packaging plasmids (and vice versa)? A1...plasmids can be packaged with either system. Please note that the generation used to package the virus does...co-transfecting three essential ingredients: the lentiviral packaging vector, a transfer vector including the gene of... an envelope. Three generations of lentiviral packaging systems have been developed over the years based...most experiments; however, the 3rd-generation packaging systems offer the maximal biosafety available ...different plasmids into the producer cells (two packaging plasmids, an envelope plasmid, and the lentiviral...and gamma-retroviruses use the same genes for packaging (that's gag, pol, and env), the isoforms of these...

  9. Addgene is Expanding Our Viral Vector Service!

    Type
    Blog Post
    ...receive your Packaged on Request prep 6–9 weeks after placing your order.  Can I order Packaged on Request...expanding our viral vector service to include a Packaged on Request option. Through this expansion, scientists...Figure 1: The process of requesting and ordering a Packaged on Request viral vector prep.    Excited? ...in the Addgene repository will be available for Packaged on Request service. A small subset of AAV plasmids...launch, we will offer five different serotypes for packaging: AAV1, AAV2, AAV5, AAV8, or AAV Retrograde (AAVrg...expansion happen?  You will be able to request Packaged on Request service starting in January 2025.  ...requestors.  Requesting Plasmids available for Packaged on Request consideration will have a banner on...

  10. Lentiviral Vector Uses and Overview

    Type
    Blog Post
    ...of three plasmids: the packaging, envelope, and transfer plasmids. The packaging plasmid carried a mutated...Advantages of lentivirus include a large genetic capacity and the ability to transduce both dividing and...mutated HIV-1 provirus that couldn’t package itself due to a few missing proteins. The envelope plasmid ...terminal repeats (LTRs) that facilitate viral packaging and host genome integration. After co-transfection...the brain (Figure 1). 2nd generation lentiviral packaging system The graphic below shows how the lentiviral... infect CD4+ cells. 3rd generation lentiviral packaging system Although the 2nd generation system is safer...virus via recombination between the transfer and packaging plasmids does exist. To further reduce this possibility...

  11. CRISPR/Cas9 FAQs Answered!

    Type
    Blog Post
    ..."CRISPR", short-hand for Clustered Regularly Interspaced Short Palindromic Repeats, in Pubmed returned...want to use double nickase, you could express two spacers and use PX335 to express the Cas9n (nickase). The...PAM sequence to the oligo when cloning my target spacers into the PX330 vector backbone? A2: There is no...usually adds it when they order the oligo. If your spacer sequence starts with a 'G', you naturally have ... compared to where the target site sequence (protospacer+PAM) is located? If there are other PAMs in the... there needs to be extensive homology of the protospacer followed by PAM. See Addgene's CRISPR resources... together, with each plasmid having a targeting spacer cloned into the backbone (pX330 or PX335, depending...

  12. University of Florida Serotype Testing Panel for the Eye and Brain

    Type
    Collection
    ... Viral Vector Packaging Service AAV University of Florida Serotype Testing...Testing Panel for the Eye and Brain Viral Vector Packaging Service: University of Florida Serotype Testing... directly to the brain parenchyma or subretinal space Serotypes The following serotypes were developed...acknowledge Shannon Boye and cite Boye, et al. 2016. Impact of Heparan Sulfate Binding on Transduction of Retina...32304666 Don’t See What You’re Looking For? Our Packaged on Request service offers you even more options...Please note this does not guarantee viral vector packaging service, but lets us know what viruses would be...

  13. Selecting Your Plasmid Purification Kit

    Type
    Blog Post
    ...cardboard packaging, there are some less obvious improvements that reduce the environmental impact. Using...waste from packaging Made from 100% post-consumer recycled material Reduced cardboard packaging Plant-based..., all kit components and packaging Yes, most kit components and packaging Yes, most kit components...to increase your output. But, exceeding a kit’s capacity can actually reduce your ultimate plasmid yield...ensure proper lysis, but you will still face column capacity limitations since the columns can be “clogged”...magnet as you scale up. Table 1: Standard Prep Kit Capacities* Prep size Amount of culture Approximate ... are looking to reduce your lab’s environmental impact, check out Addgene’s blog post on bringing sustainable...

  14. Build Your CRISPR Vocabulary

    Type
    Blog Post
    ...with alternating spacers and repeats, was the original impetus for studying CRISPR. Spacers: Copies of foreign...sequences are called protospacers. Repeats: Bacterial DNA that appears between each spacer in the CRISPR array...the CRISPR array and include a spacer and a repeat sequence. The spacer sequence is the RNA guide, like...real mouthful of a phrase: Clustered Regularly Interspaced Short Palindromic Repeats. That contrast of simplicity...composed of identical repeat sequences and variable spacer sequences. The array is transcribed and processed... into crRNAs, each including one repeat and one spacer. In bacteria, these crRNAs are bound by Cas proteins... sequence base pairs with the tracrRNA, and the spacer sequence is used to target complementary DNA sequences...

  15. Viral Vectors 101: Viral Vector Elements

    Type
    Blog Post
    ...well, though. Viral packaging and entry The genetic cargo requires viral packaging to infiltrate and integrate...from the transfer plasmid. Viral packaging and entry The viral packaging proteins for AAV are rep and cap...Addgene blog Parts of the AAV packaging plasmid Overview of Lentiviral Packaging Plasmids Adenoviral production...multiple plasmids with many components are used to package lab grown viruses. It can be intimidating at first...transfer plasmid while the other plasmids with packaging or replication components are called helper plasmids...). These LTRs define the limit of what will be packaged. The 5’ LTR and 3’ LTR, respectively, act as RNA...nucleocapsid viral components needed for viral packaging, assembly, and display. Pol, the last component...

  16. Pipetting Protocol

    Type
    Protocol
    ...to the amount you are dispensing can negatively impact your experimental results. This protocol will guide...

  17. Production of Virus in Insect Versus Mammalian Cells

    Type
    Blog Post
    ...have different packaging capacities depending on the virus. AAV has a packaging capacity of ~4.7 kb, while...containing your gene of interest plus two packaging plasmids (three packaging plasmids for 3rd generation lentivirus...Baculovirus transfer plasmids have a much larger packaging capacity — up to 38 kb! Generally, insect cell production...lentivirus production). The packaging plasmids are widely available, so you’ll only have to worry about cloning...while lentivirus has a capacity of 8–10 kb. Time Viral production in mammalian cells is relatively quick...construct, called a bacmid. Like AAV and lentiviral packaging plasmids, bacmids are readily available, so the...if needed # of plasmids Three or four Two Packaging limit ~4.7 kb (AAV); 8–10 kb (lenti) 38 kb Cells...

  18. Overcoming the AAV Size Limitation for CRISPR Delivery

    Type
    Blog Post
    ...into an AAV vector is challenging due to its packaging capacity of AAV (~4.7 kb). While this approach has...previously packaged Cas9 and multiple gRNAs into separate AAV vectors, increasing overall packaging capacity...preferential infection of certain tissues. However, packaging Streptococcus pyogenes (SpCas9) and a gRNA together... the only CRISPR enzyme that’s small enough to package into AAV. At 984 amino acids in length, Cas9 from...Cas9 (NmeCas9) at 1,082 amino acids, can also be packaged into AAV. NmeCas9 has the added advantage that...respectively, the size of both is within AAV’s packaging limit. For CRISPR-based RNA editing, the REPAIR...approximately 4.1 kb in length, allowing them to be packaged in AAV.   Split AAV approaches  While finding...

  19. Adenoviral Vector Production and Troubleshooting

    Type
    Blog Post
    ...high capacity AdV) are devoid of all viral coding sequences and retain only the ITRs and packaging signal... Ad vectors (helper-dependent (HD-Ad), or high-capacity (HC-Ad__ are devoid of viral genes and therefore...kb. Gutless Ad maintain only the ITRs and the packaging signal (Ψ), both of which are essential for assembly...such as HEK293 cells. 1st generation AdV cloning capacity is limited to 8.2 kb, and in vivo transgene expression...RCA) can be generated if the E1 gene from the packaging cell line is transferred into the AdV by recombination...Gen AdVs were designed to have increased cloning capacity, decreased  potential for the generation of RCA...2nd generation AdVs, thereby increasing cloning capacity to ~12 Kb. The ever popular pAdEasy system belongs...

  20. 5 Tips for Troubleshooting Viral Transductions

    Type
    Blog Post
    ...contaminating your transduction with the packaging cells. Packaging cell debris can be removed either by ... particles in a smaller volume. Always remove packaging cell debris before proceeding to ultracentrifugation...step (5 minutes at 300-500 g). After removing packaging cell debris, virus can be pelleted by ultracentrifugation...reduce the volume of cell culture medium on the packaging cells immediately following transfection. In other...fold (Stockschläder et al., 1999). 5. Check your packaging cells for infection If your transductions aren... virus is being generated by your transfected packaging cells. This check can be performed routinely and...disrupting the flow of your experiment. If your packaging cells have been successfully transfected and produced...
Showing: 41 - 60 of 562 results