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We narrowed to 563 results for: cat.3

Showing: 501 - 520 of 563 results

  1. Antibodies 101: Introduction to Immunofluorescence

    Type
    Blog Post
    ... remember that antibodies recognize very specific 3-dimensional epitopes. Different sample processing ...knowing that where you see fluorescence indicates your target’s location. Some other terms you might see associated...immunofluorescence? Generally speaking, IF is an application in which antibodies that have been joined to ...bind to a single primary, resulting in signal amplification. Another reason is cost effectiveness. Generating...distribute antibodies validate them in a variety of applications, including IF (or IHC or ICC), and will share...will share their findings either through their publications or as a review on a company website. It is important...Resources on the Addgene blog Learn about research applications of antibodies in Introduction to Antibodies ...

  2. Adeno-associated Viruses (AAVs) for Genome Editing

    Type
    Blog Post
    ... PMID: 14704360. PubMed Central PMCID: PMC373311. 3. Kim, Jung-Sik, et al. "Epitope tagging of endogenous...gene at once. Therefore, applications that require simultaneous modification of all alleles of a gene,... recommends designing homology arms with the modification of interest, ordering them from a gene synthesis...with a homologous repair template. It is this application - the creation of sequence-specific knockins ...clones, making it unsuitable for most in vivo applications.  Third, even in the most optimized tissue-culture...tissue culture based approaches that require modification of only a single allele of a gene, such as the...efficiently deliver large repair templates for applications that require large gene fusions. The current...

  3. Getting the Most from Your Lentiviral Transduction

    Type
    Blog Post
    ...Biotechniques 35.4 (2003). 812-4. Pubmed PMID: 14579747. 3. Drexler, H.G., Uphoff, C.C. “Mycoplasma contamination...positively charged polycation, polybrene. Interestingly, one study found that alternative polycations such as DEAE-dextran... freshly collected lentivirus for downstream applications. While this ensures that the highest titer virus...lentivirus may want to consider testing a variety of cation and serum sources to ensure the best possible transduction...establishes the best conditions for specific applications, stick with them! Changes in such variables ...efficiency of lentiviral vectors: effect of sera and polycations.” Molecular Biotechnology 53.3 (2013). 308-14...

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

    Type
    Blog Post
    ...biotechnology 16(7): 657–62. PubMed PMID: 9661200.  3. Cardin, Jessica A. et al. 2009. “Driving Fast-Spiking... gives you a single level of control without modification - either Cre is there or it’s not. Cre-mediated... sites can lead to excision, inversion, and translocation of DNA in a similar fashion as between loxP ...37 °C). Smart molecular evolution led to the identification of FLPe which has a temperature optimum of ...-LDB domains of the fusions allowing Cre/FLP translocate to the nucleus where they exert their recombinase...

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

    Type
    Blog Post
    ...visualized and sorted via flow cytometry. Figure 3: Targeting pEF-BFP with a cytidine deaminase base ...2019). TREE is a real-time, fluorescent based identification system for the isolation of base-edited cell...to BFP Conversion: A Versatile Assay for the Quantification of CRISPR/Cas9-mediated Genome Editing. Molecular...Harris RS (2018) A fluorescent reporter for quantification and enrichment of DNA editing by APOBEC–Cas9...

  6. Lentiviral Vector Guide

    Type
    Guide
    ...activation. Second-generation plasmids include (Figure 3): Transfer plasmid — contains transgene and wild-type... expression from the LTR is Tat-dependent. Figure 3: Second-generation lentiviral plasmids. Created with... transfer plasmids also contain a deletion in the 3’ LTR to make them self-inactivating (SIN). This deletion...requirement for Tat. Always includes deletion in the 3' LTR for self-inactivation LTR Viral Promoter Wildtype...) plasmids. These plasmids have a deletion in the 3' LTR of the viral genome that is transferred into .... The inside out of lentiviral vectors. Viruses , 3 (2), 132–159. https://doi.org/10.3390/v3020132 PMID...Molecular Therapy — Methods & Clinical Development , 3 , 16017. https://doi.org/10.1038/mtm.2016.17 PMID:...

  7. Molecular Cloning Techniques

    Type
    Guide
    ... interest into fragments containing precise 5' or 3' single-strand overhangs (sticky ends), or no overhang...Gateway attB1 and attB2 sites attached to the 5’ and 3’ ends of the DNA sequence. This fragment can then ...which naturally leaves a single A overhang on the 3' end of PCR products. The complementary T comes from...covalently bound to the phosphate group on the free 3' T. The topoisomerase acts as a ligase that joins ... Read more in our TOPO Cloning blog post . Figure 3: Summary of TOPO cloning. TOPO = topoisomerase I. ... Ligation Independent Cloning (LIC) relies on the 3' to 5' exonuclease activity of T4 DNA polymerase. ... Educational Resources Molecular Biology Reference Molecular...

  8. Gamma-Retroviral Vector Guide

    Type
    Guide
    ...Gammaretroviral vectors: Biology, technology and application. Viruses , 3 (6), 677–713. https://doi.org/10.3390/...enables additional flexibility in pseudotyping. Figure 3: Gamma-retroviral vector packaging methods. Created...self-inactivating (SIN). These plasmids have a deletion in the 3' LTR of the viral genome that is transferred into ...found on each side of the provirus. The U3 (unique 3’) contains sequences necessary for activation of viral...transcription. R is the repeat region. U3 in cis Unique 3’; contains sequences necessary for activation of viral... RNA transcription. Removal of this region in the 3’ LTR creates self-inactivating viral vectors. R in... with a constitutive promoter such as CMV or RSV. 3' LTR in cis Terminates transcription started by 5'...

  9. Plasmids 101: Biotinylation

    Type
    Blog Post
    ...Sciences, 24.9 (1999b): 359–363. PubMed PMID: 10470036. 3. Said, H. M. “Biotin: The forgotten vitamin.” American...molecular biology applications for biotin and avidin. A short list of some other common applications and links...downstream application. Biotin labelled oligonucleotides can be used in a multitude of applications including... carboxylase and decarboxylases. These enzymes catalyze the transfer of carboxyl groups to organic acids...this highly specific post-translational protein modification. Most organisms have less than five enzymes ...uses of biotinylation in the lab are protein purification and immunoprecipitation experiments. Biotinylated...sites found on avidin are also ideal for signal amplification during these experiments. During detection, ...

  10. Transferable Skills Guide: Conflict Resolution

    Type
    Blog Post
    ...thomas-kilmann-conflict-resolution-model/, accessed Jan. 3, 2022.     Additional Resources on the Addgene blog...conflict is not always what was communicated but how it was communicated. The next step is to come up with...of lapsed communication. Making some assumptions about how something should be communicated is necessary...skill in and of itself. Conflict can also be categorized as either “personal” or “substantive”. A personality-based...traced back to one of a few trouble spots:  Communication (or lack thereof): Information sharing gets ...organization gets, and creating new avenues of communication can create as many problems as it can solve....meeting? Different teams frequently prefer to communicate in different ways, so the source of the conflict...

  11. Finding nucleic acids with SHERLOCK and DETECTR

    Type
    Blog Post
    ...recognizes a different PAM site and generates 5’ and 3’ staggered ends after dsDNA breaks. The Doudna lab...activated” Cas13 produces a quantifiable signal that indicates the presence of your targeted nucleic acid. To...amplified using RPA (recombinase polymerase amplification) or reverse transcriptase (RT)-RPA, respectively...RNA for subsequent detection by Cas13. This amplification step in combination with the ssRNA reporter ... introduced in 2017 so the lab made further modifications to improve their detection system. They named... SHERLOCKv2 uses far less primer in the pre-amplification step allowing for greater quantitation without...hour. Find the SHERLOCK plasmids at Addgene! Applications of the SHERLOCK detection system The Zhang lab...

  12. Fluorescent Proteins 101: GFP Fusion Proteins - Making the Right Connection

    Type
    Blog Post
    ...predict which one will work as illustrated in figure 3 (Mastop et al., 2018). Unbiased, random insertions...study its function is one of the most popular applications of fluorescent proteins. These fusion proteins...steric hindrance. For instance, GFP may occlude catalytic sites or obstruct binding sites or motifs that...that are necessary for post-translational modifications. Despite these potential issues, GFP has been used...linker sequence short In the early days of GFP applications, many were concerned with steric hindrance by...N-terminus of the donor fluorescent protein can be truncated to increase the FRET efficiency. Creating fusions...fluorescent protein and protein of interest (POI). To indicate the sequence, people often use terminology like...

  13. Adeno-associated virus (AAV) Guide

    Type
    Guide
    ...splice donor sequence at its 3' end, while the second vector carries the 3' portion of the gene with a ...Rep proteins essential for viral DNA replication and packaging. 3' ITR (RITR) in cis Right Inverted Terminal...and promotes RNA transcript longevity. Located between WPRE and 3'ITR. Not required but often included....protocols, see Addgene’s Viral Vector Protocols . Figure 3: Overview of rAAV vector production. Created with ...frequency which has been estimated to range from 0.01% to 3% depending on cell type and rAAV vector concentration...vectors for human gene therapy . Current Gene Therapy, 3 (4), 281–304. https://doi.org/10.2174/1566523034578285...enhanced gene transfer . Pharmaceutical Research, 25 (3), 489–499. https://doi.org/10.1007/s11095-007-9431...

  14. Plasmids 101: Gibson Assembly and Other Long-Homology Based Cloning Methods

    Type
    Blog Post
    ...BioTechniques 2015; 58:161-170. PubMed PMID: 25861928. 3. Jiang W, Zhao X, Gabrieli T, Lou C, Ebenstein Y, ...neighboring fragments can be created via PCR amplification with primers that contain the appropriate homologous... tools that help you design primers for PCR amplification of fragments to incorporate such regions of ...assembly meets CRISPR Gibson can be adapted to more complicated cloning schemes, such as those where the vector...CRISPR technology and is described in a recent publication by the Lockey lab (Wang, et al. 2015). In this...aspects of the SLIC methods. First, it uses a dedicated 5’ exonuclease instead of using the exonuclease...DNA fragments contain these sites at the correct location and NOT in the middle of your insert. Gateway ...

  15. Viral Vectors 101: Integrase-Deficient Lentiviral Vectors

    Type
    Blog Post
    ...without the risks associated with prolonged expression. 3. Stable expression in non-dividing cells IDLVs can...decades. Widely used across various research applications, they have become indispensable tools for manipulating... easily, without the need for time-consuming purification processes, and are powerful, due to their ability...-lasting changes in cells. However, for some applications, integration might be considered a double-edged...dividing cells because they lack an origin of replication (Banasik and McCray, 2010). However, IDLV genomes... certain types of clinical or basic research applications. Considerations for IDLV use If you’re reading...Integrase-defective lentiviral vectors: progress and applications. Gene Therapy, 17(2), 150–157. https://doi.org...

  16. CRISPR 101: Any Base Transversion Editors

    Type
    Blog Post
    ...base editor development going forward. Figure 3: Mechanism of gGBE. Created with BioRender.com. ...Red arrows indicate a conversion directly catalyzed by the base editor. Black arrows indicate natural cellular...researchers can now select the best tool for their application from among many options.  Conclusion With these...engineered human uracil DNA glycosylase. Nature Communications, 15(1), 4897. https://doi.org/10.1038/s41467...via engineered uracil-DNA glycosylase. Nature Communications, 15(1), 6397. https://doi.org/10.1038/s41467...

  17. Five Popular Model Organisms, Part 2

    Type
    Blog Post
    ...Model for Regeneration and Aging. Gerontology, 66(3), 212–222. https://doi.org/10.1159/000504294 Wang,...studying genetic modification for their ease of manipulation and their ability to be replicated. Because E...., 2004). Additionally, breakthroughs in gene modification tools and methods such as CRISPR have vastly...Escherichia coli as a Model Organism and Its Application in Biotechnology. In Escherichia coli - Recent...Physiology, Pathogenesis and Biotechnological Applications. InTech. https://doi.org/10.5772/67306 Koornneef... bacteria with superior performance. Nature Communications, 9(1). https://doi.org/10.1038/s41467-018-04899...

  18. Fluorescent Proteins 101: When GFP lets you down

    Type
    Blog Post
    ...protein." (1998): 509-544. PubMed PMID: 9759496.  3. Buckley, Anthony M., et al. "LOV-based reporters ... proteins.Therefore, GFP is not suitable for applications that require anaerobic conditions. In experiments...an additional protein module that can report on location. This approach enables the study of proteins in...fusion with GFP allows for detection of genomic locations (Chen et al., 2013). GFP fluorescence overlaps...case when proteins undergo post-translational modification with fatty acyl chains. For instance, myristoylation...C-terminal consensus sequence (-CaaX). Both modifications are disrupted when GFP is fused to these sequences...

  19. Plasmids 101: Common Lab E. coli Strains

    Type
    Blog Post
    ...strain. F- Δ(ara-leu)7697 [araD139]B/r Δ(codB-lacI)3 galK16 galE15 λ- e14- mcrA0 relA1 rpsL150(strR) spoT1...that carried out the labor-intensive process of replicating your new plasmid: the Escherichia coli bacteria...conjugation. Genes listed on F´ are wild-type unless indicated otherwise fhuA (formerly tonA) ferric hydroxamate...vectors. Used by Addgene for pooled library amplification. F' mcrA Δ(mcrBC-hsdRMS-mrr) recA1 endA1 gyrA96...typically standard; just having the gene listed indicates it is non-functional. If a gene is deleted, it... Most Popular Plasmids 101 Posts: Origin of Replication The Promoter Region – Let's Go! Mammalian Vectors...

  20. Promoters

    Type
    Guide
    ...example of a negatively-controlled operon (Figure 3). A positive-repressible operon works in the opposite...example of a positively-controlled operon. Figure 3: Lac operon regulation. Created with BioRender.com...proteins) make up the transcription complex. The most 3' portion (closest to the gene's start codon) of the...Constitutive Yeast Very strong promoter from glyceraldehyde 3-phosphage dehydrogenase; also called TDH3 or GAPDH... Educational Resources Molecular Biology Reference Promoters...BioRender.com. DNA sequences called response elements are located within promoter regions, and they provide a stable...Promoters in bacteria contain two short DNA sequences located at the -10 (10 bp 5' or upstream) and -35 positions...
Showing: 501 - 520 of 563 results