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We narrowed to 98 results for: plasmid transfection

Showing: 21 - 40 of 98 results

  1. Google Forums Round Up: First Impressions of NgAgo

    Type
    Blog Post
    Published
    Aug. 4, 2016, 2:30 p.m.
    ...researchers must co-transfect cells with 5’-P-ssDNA guides and an NgAgo expression plasmid to edit a gene ...used by respondents (transfection method, amount of each component per transfection etc.). Take Pooran'...sequencing on select regions of all distributed plasmids as part of quality control, but does not perform...unlike an RNA guide, it cannot be produced from a plasmid in cells. Additionally, in vitro assembly of NgAgo...delivery methods. Researchers can conveniently transfect/inject cells with in vitro preassembled Cas9/gRNA...complexes or with guide RNA- and Cas9-expression plasmids. Cas9 guide RNA delivery does however rely on ...(1), NgAgo has received much attention and the plasmid has been requested by labs around the globe nearly...

  2. Viral Vectors 101: Producing Your rAAV

    Type
    Blog Post
    Published
    July 16, 2024, 1:15 p.m.
    ...packaging plasmids: the pHelper plasmid containing Ad E2A, E4, and VA RNA genes; and the pRepCap plasmid containing...interest, and the two helper plasmids, commonly called “triple plasmid transfection” (Figure 1).    Figure ...expressing Ad E1A and E1B, can be transfected with your transfer plasmid, carrying your gene of interest... performed 12–18 hours post-transfection to help remove toxic transfection reagents and keep the cells...cells happy. Harvesting virus Once you have transfected your cells, wait 2–5 days and then collect the supernatant...repeats (ITR). You should always have a high-quality plasmid standard and rAAV reference material of a known...before undergoing titering, to remove residual plasmid DNA carried over from the production process. For...

  3. Antibodies 101: Validation

    Type
    Blog Post
    Published
    March 24, 2022, 1:15 p.m.
    ...protein of interest endogenously and transfect them with a plasmid encoding your protein of interest. Then...acquire a plasmid encoding your protein of interest, known as Gene of Interest (GOI), and transfect it into...favorite cell line. In parallel, you transfect cells with another plasmid not encoding this protein, which... the faintest band from the cells transfected with the other plasmid. Your antibody works! Multiple (Independent...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...Antibodies blog post!  Resources on Addgene.org Find plasmids encoding antibodies at Addgene Stay tuned for ...

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

    Type
    Blog Post
    Published
    Oct. 23, 2018, 11:49 a.m.
    ... is transfecting HEK293 cells with AAV packaging plasmids. The cells need three different plasmids to ...AAV plasmid standard and AAV transfer plasmids. It’s easy to contaminate a workspace with plasmid, not...going to lie, AAV transfer plasmids are kind of a pain to work with. These plasmids contain ITRs which are...packaging PEI transfections. It saves me time and helps me figure out if I have enough plasmid to complete...to produce AAV: 1) the RepCap plasmid which provides the AAV replication (rep) and capsid (cap) genes. ...AAV’s protein shell, or capsid; 2) the pHelper plasmid which expresses adenovirus genes which help mediate...mediate AAV viral replication; and 3) the transfer plasmid which encodes a transgene of interest that’s packaged...

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

    Type
    Blog Post
    Published
    Sept. 26, 2019, 12:50 p.m.
    ...cells. Options for CRISPR delivery include plasmid transfection, RNP electroporation, and viral transduction...effects when compared to DNA transfection of gRNA and Cas9 expression plasmids. These results are in line... non-replicative. Nanoblades are produced by transfecting the following four components into 293T cells...types they tested. Interested in using Nanoblades? Plasmids are available from Addgene. Genome editing primary...having lower off-target edits compared to DNA transfection.  Homology directed repair (HDR) with Nanoblades...out our comprehensive CRISPR guide Find CRISPR plasmids and resources Learn about viral production ...

  6. Viral Vectors 101: Viral Applications

    Type
    Blog Post
    Published
    April 13, 2023, 1:15 p.m.
    ...higher than traditional transfection and random genomic integration of a plasmid. Viral transduction is...sequence for degradation. Viral introduction of shRNA plasmids can induce gene-specific silencing temporarily...due to the ease of transduction in difficult-to-transfect cell types ranging from primary to embryonic stem...is also generally less toxic to cells than transfections, preserving cell viability. Library delivery... with viral vectors instead of a traditional transfection method. This is because viral systems have a...

  7. PiggyBac-ing Through the Genome Editing Field

    Type
    Blog Post
    Published
    May 31, 2016, 3:30 p.m.
    ...found that transfecting with PBx mRNA can boost efficiency over transfecting with plasmids in some cell...the ability to distribute plasmids with the piggyBac™ transposon. These plasmids, when combined with a source...fully intact, while, with a standard DNA plasmid transfection, a double-stranded break may occur in your...useful resource. While Addgene cannot distribute plasmids with the piggyBac™ transposase itself, please ...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...by utilizing a piggyBac™ transposon in a donor plasmid carrying the DNA edit of interest, along with a...

  8. New Acoustic Reporter Genes: Ultrasound Imaging of Gene Expression

    Type
    Blog Post
    Published
    Jan. 24, 2023, 2:15 p.m.
    ...that the plasmid could be maintained in the absence of antibiotics, resulting in the plasmid pBAD-bARGSer-AxeTxe... accessory proteins. Excess gvpA plasmid added into the transfection mixture in HEK293T cells gave drastic...with Emerald GFP. These plasmids can be used directly in transient transfection in rtTA+ cell lines (such... 2 after transfection.     Fig: 2: Expressing mARGAna in MDA-MB-231 cells. (a) Plasmid diagram ... be integrated into the genome by cotransfecting them with a plasmid expressing PiggyBac transposase. ...transient cotransfection of the mARG plamsids into HEK293T cells using a chemical transfection reagent ...e.g. PEI-MAX). Simply cotransfect a mixture of 1248 ng of CMV-gvpA-IRES-BFP plasmid and 954 ng CMV-gvpNJKFGWV...

  9. Advanced Uses of Cre-lox and Flp-FRT - A Neuroscientist’s View

    Type
    Blog Post
    Published
    Oct. 19, 2017, 1:54 p.m.
    ....org Find Cre-Lox Plasmids Find Optogenetics Plasmids Find Chemogenetics Plasmids ...individual neurons one could reduce the plasmids amounts to reduce transfection efficiency. However, if the cellular... guarantees that most cells are transfected with all labeling plasmids and the low concentration of recombinase...vectors expressing light inducible ion channels (plasmid # 20298 for example) have revolutionized Neuroscience...neurons in the living brain   Figure 1: Plasmid mix to label neuronal morphology (eYFP) and the...recombinase. After in-utero electroporation, this plasmid mix results in sparse labeling of cortical neurons...brain? Cells of a specific brain area can be transfected by in utero electroporation. To achieve sparse...

  10. Fluorescent CRISPR Reporters: SRIRACCHA and GEmCherry2

    Type
    Blog Post
    Published
    May 26, 2020, 1:15 p.m.
    ...simply changing the gRNA used when transfecting your cells.  A plasmid that contains Cas9. The Madison lab...control RFP expression plasmid.  With this set up, all successfully transfected cells will express RFP..., you’re ready to edit. These cells are then transfected with several different constructs for the reporter...and to make the desired genome edit:  A donor plasmid that contains a puromycin resistance gene linked... and repaired via HDR using the supplied donor plasmid will express GFP. If there’s GFP expression, presumably...just your desired genomic edit.  Find SRIRACCHA plasmids at Addgene GEMCherry2, an improved monomeric fluorescent...embryonic stem cells.  Find GEmCherry2 reporter plasmids at Addgene Overall SRIRACHA and GEmCherry2 reporter...

  11. Plasmids 101: Protein Expression

    Type
    Blog Post
    Published
    June 7, 2018, 1:17 p.m.
    ...modifications Learn more molecular biology in our Plasmids 101 eBook! Researchers use various techniques ... strategy for protein expression consists of transfecting cells with your DNA template of choice and allowing...SV40, CMV, and RSV) for robust expression post-transfection. Mammalian systems can express proteins both... Both methods produce high protein yields if transfection is successful. Some mammalian systems also ...for your protein of interest is inserted into a plasmid expression vector that is then transformed into...being able to help the scientific community share plasmids.   Additional Resources Thermofisher Protein ...Addgene.org Bacterial expression systems Search for plasmids by expression system ...

  12. Your Lentiviral Plasmid FAQs Answered

    Type
    Blog Post
    Published
    April 23, 2014, 1:08 p.m.
    ...involves transfecting four different plasmids into the producer cells (two packaging plasmids, an envelope...generation systems consist of an envelope plasmid and a packaging plasmid encoding HIV proteins gag, pol, rev...generation systems require an envelope plasmid, and two packaging plasmids, one encoding gag and pol and a second...transfer plasmids replication competent? What is SIN? A6: Most (if not all) Addgene transfer plasmids are ... 30% of the transfer plasmid. Many transfer vectors are SIN. Packaging plasmid(s) contain the minimal...Addgene's most popular lentiviral plasmids Overview of Lentiviral Packaging Plasmids Lentiviral Protocols and ...Addgene has an extensive collection of lentiviral plasmids created for a variety of applications including...

  13. Viral Vectors 101: Preparing Pooled Libraries

    Type
    Blog Post
    Published
    July 13, 2023, 1:15 p.m.
    ...representation. Transfection of the DNA pooled library and appropriate packaging and envelope plasmids into HEK...single plasmid transformation, though, a pooled library has hundreds to millions of distinct plasmids, all...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...Addgene.) This small aliquot is not enough to transfect producer cells, so your first step will be to ...amplify the library. This is similar to amplifying a plasmid: transform E. coli with the pooled library DNA,...

  14. Choosing Your Perfect Empty Backbone

    Type
    Blog Post
    Published
    Aug. 19, 2014, 3:39 p.m.
    ...you find what you need.   More Plasmid Resources from Addgene: Plasmids 101 blog series Addgene's Molecular...need. You will also have to make sure that your plasmid has been incorporated into the host organism, usually...mammalian selection cassette to screen for positive transfected cells. The addition of an in-frame fluorescent...you can get away with generating transiently transfected cells, you will not need a mammalian selection...open reading frame (ORF) of interest –  try pRS plasmid shuttle vectors. You may prefer the more traditional...details and guidance for choosing your perfect plasmid backbone. Or just ask –  leave a message in the...Molecular Cloning Guide & Other Plasmid Protocols List of common epitope tags ...

  16. What the HEK?

    Type
    Blog Post
    Published
    April 18, 2023, 1:15 p.m.
    ...viral plasmids with the SV40 origin of replication. To generate a virus, you need only transfect the ‘... ‘helper’ plasmids (viral packaging machinery) along with the viral plasmid containing the cargo of interest...Protein production  HEKs are extremely easy to transfect in bulk and produce recombinant proteins at high...

  17. Viral Vectors 101: Gamma-Retroviral Packaging Systems

    Type
    Blog Post
    Published
    Aug. 6, 2024, 1:15 p.m.
    ...γ-retroviral packaging plasmids are not interchangeable. General envelope plasmids, such as VSV-G (Vesicular...in trans by specific plasmids. This method, therefore, reduces the number of plasmids that are required ...encoded by the transfer plasmid, but instead are provided in trans by other plasmids or the packaging cell...do many γ-retroviral plasmids have similar names? The nomenclature of the plasmid backbone is meant to...transfer) are supplied by three types of plasmids, which are cotransfected into the 293T packaging cell line....components are needed: γ-Retroviral transfer plasmid encoding a transgene, sgRNA, or shRNA of interest..., which facilitate integration of the transfer plasmid sequences into the host genome. Typically, it is...

  18. Four Base Editing Reporters to Monitor and Enrich Editing in Real-time

    Type
    Blog Post
    Published
    July 7, 2020, 1:15 p.m.
    ...solely by a reporter of transfection, which only report the efficiency of plasmid delivery to a cell. In...constitutively expressed to identify successfully transfected cells. C-to-T editing by the APOBEC-Cas9 editosome...In contrast, TREE provides a read out for plasmid delivery and subsequent base editing efficiency. TLDR...frequency of base edits of interest than solely transfected cells. “Gene On” (GO)- a functional reporter ...Addgene.org Browse the CRISPR guide Find CRISPR plasmids for your research ...

  19. Plasmids for Endogenous Gene Tagging in Human Cells

    Type
    Blog Post
    Published
    April 6, 2017, 1:02 p.m.
    ...Designing the donor plasmid Figure 2: Donor plasmid design strategy. A donor plasmid contains 1 kb homology...the plasmid to prevent the plasmid from being cut and destroyed by CRISPR/Cas9 during transfection. Making... the tag. Created with BioRender.com. Plasmids, plasmids everywhere To find out more about gene edited...expensive for many labs. We’ve created and tested plasmids that use CRISPR/Cas9 to endogenously tag a wide...wide variety of genes with GFP. These plasmids are available through Addgene, and the stem cell lines we...found in the Cell Catalog on our data portal. The plasmids should be functional in many different human cell...constructs below. Read on to find out how these plasmids work and to discover more resources for your genomic...

  20. Tips for Titering Your Lentiviral Preps

    Type
    Blog Post
    Published
    March 15, 2016, 2:30 p.m.
    ...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 ...for expression of a gene carried on the transfer plasmid or quantifying the number of viral copies that ... that comes from amplification of the original plasmid which can over-inflate results. A packing vector...antibiotic resistance genes carried on transfer plasmids. Target cells are transduced with serial dilutions...
Showing: 21 - 40 of 98 results