Skip to main content
Addgene

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

In Category

We narrowed to 393 results for: ara-2

Showing: 21 - 40 of 393 results

  1. Viral Vectors 101: Producing Your rAAV

    Type
    Blog Post
    Published
    July 16, 2024, 1:15 p.m.
    ...digital PCR. Human Gene Therapy Methods, 25(2), Article 2. https://doi.org/10.1089/hgtb.2013.131 Lock,...Snyder, R. O. (2010). Characterization of a recombinant adeno-associated virus type 2 Reference Standard ... virus Once you have transfected your cells, wait 2–5 days and then collect the supernatant and/or producer...quantification of adeno-associated virus serotype 2-derived inverted terminal repeat sequences. Human ..., R., & Kleinschmidt, J. (1999). Titration of AAV-2 particles via a novel capsid ELISA: Packaging of genomes... genomes can limit production of recombinant AAV-2. Gene Therapy, 6(7), Article 7. https://doi.org/10.1038...been developed to determine the titers of rAAV preparations, such as dot blot, enzyme-linked immunosorbent...

  2. Plasmids 101: Secondary Nanobody Toolbox

    Type
    Blog Post
    Published
    Feb. 27, 2018, 2:04 p.m.
    ... larger label displacement Require separate incubation of 1° and 2° Must use different species and/or...antibody has an antigen binding site for a total of 2 binding sites per antibody. C) IgG’s are big enough...enough that multiple labels (green circles in figure 2 below) can be attached to each antibody. But being...Greater resolution due to lower label displacement 2° incubation can be skipped by pre-binding to 1° ...a fluorescent tag and the target antigen to about 2 nm, which can lead to higher resolution for super-...subclass.   Express the Nanobody In Bacteria: Table 2 also has a link to the plasmid you’ll need to express... Find plasmids from this publication at Addgene. 2. Bates, M., Böhning, M.I., Chug, H., Görlich, D., ...

  3. Five Popular Model Organisms, Part 2

    Type
    Blog Post
    Published
    Sept. 29, 2020, 1:15 p.m.
    ... H. J. (2009). Rats! Disease Models & Mechanisms, 2(5–6), 206–210. https://doi.org/10.1242/dmm.002733 ...is as a biomedical model organism.  Arabidopsis thaliana Arabidopsis thaliana is one of, if not the most...scientific history, highly characterized genome, and widespread utilization, Arabidopsis has made significant...community.  The Arabidopsis genome project marked the beginning of widespread Arabidopsis genetics research...world worked to characterize each gene’s function. Some key genetics research with Arabidopsis include understanding...feature genetic, physiological, or developmental characteristics that make them ideal for studying in a laboratory...genome has an extensive research history and characterization. Many proteins and processes naturally found...

  4. Adenoviral Vector Production and Troubleshooting

    Type
    Blog Post
    Published
    June 14, 2018, 11:44 a.m.
    ...repeated multiple times (2-4 rounds) at increasing scale over the course of 1-2 weeks. Each round of amplification...creating adenoviral vector constructs. It consists of 2 plasmids: a shuttle vector (in which the transgene...Addgene’s Adenoviral Guide webpage, and references 1, 2.*Pro-Tip*: once the correct recombinant pAdV plasmid...sequenced to confirm its integrity. Initial production (2-3 weeks) - Here you’ll produce the primary recombinant...media will turn yellow. Do NOT change the media (add 2-3 mL of fresh media once a week), and do NOT harvest...it will result in very low titer. Amplification (1-2 weeks) - The rAdV-S is used to infect more HEK293 ...in a 10-100-fold increase in virus. Purification (2 days) - Purification is required if rAdV is to be ...

  5. Deep Mutational Scanning with One Pot Saturation Mutagenesis

    Type
    Blog Post
    Published
    Feb. 22, 2017, 3:30 p.m.
    ... that results in a polymerase error rate of up to 2% per nucleotide position. The “sloppy” PCR reaction...modifications: 1) increased concentration of Taq polymerase, 2) increased PCR extension time, 3) increased concentration...to synthesize a complementary mutant strand (step 2 and 3). Then the uracil-labeled template strand is...restrict site matter? Most of the time no, but there are 2 instances where it does. 1) When the backbone vector...ExoIII. See the below figure for an example like this. 2) When your gene of interest has a BbvCI site(s). If...saturation mutagenesis method are 1) ease of use, 2) time savings, and 3) ability to perform single site...PMID: 23766324. PubMed Central PMCID: PMC4409680. 2. Mccullum, E. O., Williams, B. A., Zhang, J., & Chaput...

  6. Cpf1: A New Tool for CRISPR Genome Editing

    Type
    Blog Post
    Published
    Oct. 14, 2015, 2:30 p.m.
    ...Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Figure from Zetsche et al., 2015...2015. How was Cpf1 found and tested? Class 2 CRISPR systems, including the type II Cas9-based system...employed by class 1 systems. A putative new class 2 nuclease, Cpf1 (CRISPR from Prevotella and Francisella... the 3’ end of the target sequence. Figure 2: Effects of (A) double and (B) single base pair gRNA-target...Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system." Cell (2015). PubMed PMID: 26422227... Find plasmids from this publication at Addgene. 2. Zetsche, Bernd, et al. "Multiplex Gene Editing by...al. added to the CRISPR toolbox with their characterization of two Cpf1 orthologs that display cleavage...

  7. AAV Titers: Where Do They Come From And What Do They Mean?

    Type
    Blog Post
    Published
    Nov. 15, 2017, 3 p.m.
    ...PubMed Central PMCID: PMC3640491. 2. Grimm, D., et al. "Titration of AAV-2 particles via a novel capsid ELISA...Lock, Martin, et al. "Characterization of a recombinant adeno-associated virus type 2 Reference Standard ...report the physical titer of a viral solution (Figure 2). Because physical titers are used for dosing purposes...mean and how they can be compared.   Figure 2: Label that comes on Addgene’s AAV aliquots. Our titer...of genomes can limit production of recombinant AAV-2." Gene therapy 6.7 (1999). PubMed PMID: 10455443. ...methods measure different characteristics of the solution. These characteristics give us information on ...vector preparations. Addgene has used this method to generally validate our viral vector preparation protocol...

  8. Multiplexed Capture of Promoter-enhancer 3D Chromatin Structures Using CRISPR

    Type
    Blog Post
    Published
    July 2, 2020, 12:40 p.m.
    ...) FLAG-biotin-tagged dCas9, 2) BirA, and 3) one or more gRNAs Two separate lentiviruses delivering 1) ...Originally published Dec 7, 2017 and updated Jul 2, 2020. Promoters may be the star of gene regulation...sequence targeted in the experiment. Figure 2: Comparing CAPTURE and CAPTURE 2.0.     Original...) a BioTAP-tagged dCas9 and an eGFP tag, and 2) gRNAs Number of loci targeted One Many Sample type...identified and the level of on-target enrichment. 2. Determining the spatial and hierarchical organization...Cytogenetics, 11. https://doi.org/10.1186/s13039-018-0368-2  Liu X, Zhang Y, Chen Y, Li M, Zhou F, Li K, Cao H...chromatin interactions at a promoter in the well-characterized beta globin locus. The two methods had similar...

  9. Minigenomes - a Safe Way to Study Dangerous Viruses Like the Ebola Virus

    Type
    Blog Post
    Published
    March 17, 2016, 2:30 p.m.
    ...study aspects of the EBOV replication cycle under BSL-2 conditions (4). What is a minigenome?   The EBOV ...transcription by the EBOV polymerase (reviewed in 2, 5). The minigenome is capable of being replicated...nonsegmented negative sense RNA viruses (reviewed in 2, 3). Other important components of the Zaire ebola...as it would for a full-length viral genome (Figure 2). While it is technically possible to encode all four...500 ng NP, 500 ng VP35, 100 ng VP30, 100 ng L, and 2 μg minigenome plasmid per well (in a 6 well plate ...PubMed PMID: 9847328. PubMed Central PMCID: PMC103829. 2. Conzelmann, K. K. "Reverse genetics of mononegavirales... were encoded in separate (and thus smaller) plasmids. Finally, having four separate plasmids allows researchers...

  10. Fluorescent Proteins 101: Photoactivatable Fluorescent Proteins

    Type
    Blog Post
    Published
    April 25, 2017, 2:30 p.m.
    ...fluorescent state make them useful for 2-color imaging with green PA-FPs. Table 2: Properties of Selected Reversible... conventional FP imaging. PA-FPs are divided into 2 main categories - those with irreversible and reversible... forms of the chromophore which contribute to the 2 peaks of wtGFP’s excitation spectrum- a major 397 ... / N99K / S148L / I165V / Q167P / L169V / I203R) (2) and PA-mRFP1(S146H / I161V / I197H) (3) were derived...with two distinct wavelengths of light (see Table 2). The best known reversible PA-FP is the naturally... above background. The majority of PA-FPs come in 2 flavours- monomeric and tetrameric. Tetrameric PA-...most PA-FPs, but, in 2015, the Looger Lab designed 2 variants of EosFP that can withstand these conditions...

  11. CRISPR 101: Multiplex Expression of gRNAs

    Type
    Blog Post
    Published
    Sept. 10, 2020, 11:45 a.m.
    ...illustrated in the schematic below.   Figure 2: gRNA target sequences (colored rectangles) are cloned... plasmids: This plasmid set allows you to express 2-4 gRNAs, with four being the ideal number. First you...the total number of gRNAs you wish to clone, from 2-7. For example, if you’re expressing 4 gRNAs, you’...gRNAs into spectinomycin-resistant plasmids pX330S-2 to pX330S-(last gRNA number). The 5’ most gRNA is ...Multiplexing Plasmids: These plasmids allow you to assemble 2-4 gRNAs through Golden Gate or Gibson Assembly. gRNAs...Multiplex Plasmids: These plasmids allow expression of 2-5 gRNAs in zebrafish. Custom destination vectors are...and a subsequent curing protocol that requires only 2-3 hours incubation. Kondo Lab multiplexed base editing...

  12. Plasmids 101: Modular Cloning

    Type
    Blog Post
    Published
    March 12, 2024, 1:15 p.m.
    ...TUs. Level 2: Assembly of multiple Level 1 TUs into multigene constructs. Figure 2: The three ...can be queried by testing any number in the Level 2 assembly. These pathways can require a lot of optimization... of multigene constructs. PLoS One. 2011 Feb 18;6(2):e16765. doi: 10.1371/journal.pone.0016765.  Hahn,... (2020). https://doi.org/10.1186/s12870-020-02388-2 Resources on Addgene.org Addgene’s Modular Cloning...with characterized behaviors are available from Addgene and other sources; second, characterizing a part...specific functions. Most individual parts have characterized activities with predictable behaviors. While...part in different contexts and plug different characterized parts into a TU! What is modular cloning useful...

  13. Viral Vectors 101: The Retroviral Lifecycle

    Type
    Blog Post
    Published
    July 27, 2023, 1:15 p.m.
    ...that have been developed into retroviral vectors and 2.) a subclass of retroviruses called the lentiviruses...effectively. Retroviral structure   Figure 2: Structure of a retrovirus. Image re-used from Dong...Dong & Kantor, 2021 under CC-BY license. Figure 2 gives a detailed view of the structure of a retroviral...transfer of RT, which would actually occur in step 2 during uncoating. After uncoating, reverse transcription...one of the two strands’ DNA counterparts from step 2 will be integrated by the viral integrase protein ... Air. Environmental Science & Technology Letters, 2(4), 84. https://doi.org/10.1021/ACS.ESTLETT.5B00050.... But it’s simple. The RT protein is made by a separate set of instructions in the same producer cells...

  14. Antibodies 101: Designing Your First Flow Panel

    Type
    Blog Post
    Published
    May 28, 2024, 1:45 p.m.
    .... To understand why that is, let's look at Figure 2. The blue laser excites at a wavelength of 488 nm ... of PE's excitation spectrum (dotted line, Figure 2). The yellow/green laser, however, excites at 561 ... stronger — or brighter — signal.   Figure 2: Excitation and emission spectra for PE. The excitation...this example, I will describe a basic panel (Table 2, Figure 5) for a hypothetical experiment in the lab...efficiency (eGFP expression).     The first item in Table 2 is a standard and should always be included—the live...from the other fluorophores to avoid bleeding and 2) BV421 is a very bright fluorophore, which makes it...case the CD45 expression turns out to be weak. Table 2: A simple flow panel to detect live, transduced, and...

  15. Starter guide to induced pluripotent stem cells (iPSCs) part 1:  A renaissance in regenerative medicine

    Type
    Blog Post
    Published
    Oct. 30, 2018, 1:04 p.m.
    ...them in Regenerative Medicine. Acta Naturae, 2010. 2(2): p. 18-28. PubMed PMID: 22649638. PubMed Central...capability to differentiate into specialized cell types [2]. We general speak of 4 levels of stem cell potency...zygote through to the morula stage are totipotent [2-4]. Pluripotency: When a cell has the ability to differentiate...stem cells (ESCs), which I discuss in detail below [2]. Multipotency: When a cell can differentiate into...PMID: 26726264. PubMed Central PMCID: PMC4686367. 2. Jaenisch, R. and R. Young, Stem cells, the molecular...promising therapeutic tools. The two unique characteristics that define stem cells are: Their ability...disadvantages. Here is a table listing some of them: Characteristic ESC ASC Self-renewal High Low once isolated...

  16. CRISPRainbow and Genome Visualization

    Type
    Blog Post
    Published
    Feb. 28, 2017, 3:30 p.m.
    ...pCRISPRainbow-DONOR1, with dCas9 supplied by a separate vector. Figure 2: CRISPRainbow generates 6 unique colors...two genes over time, at a range of distances from 2-75 Mbp. Labeling multiple, intrachromosomal loci Building...successfully resolved loci with physical map distances of 2 and 75 Mbp, with the calculated fluorescent distances...established physical map. In comparing pairs of targets ~2 Mbp apart, they noticed that they could evaluate the... Find plasmids from this publication at Addgene. 2. Ma, Hanhui, et al. “Multicolor CRISPR labeling of... labeling efficiency was similar to that of a comparable FISH method. Importantly, they did not observe...intrachromosomal loci, a major benchmark in characterizing the 4D nucleome. CRISPRainbow: Scaling up to...

  17. Typing CRISPR Systems

    Type
    Blog Post
    Published
    March 18, 2025, 1:15 p.m.
    ...   Class 1 versus class 2 There are two CRISPR classes: Class 1 and Class 2. Class 1 CRISPR systems use...systems to carry out various functions. Class 2 Class 2 CRISPR systems rely on a single protein Cas effector...explore Class 2 CRISPR in more depth, the Jennifer Doudna lab maintains an encyclopedia of Class 2 CRISPR systems...systems: a burst of class 2 and derived variants. Nature Reviews Microbiology, 18(2), 67–83. https://doi.org...complex for antiviral defense) complexes, while Class 2 use single protein Cas effectors, like Cas9. The Cascade...1 remains largely underutilized compared to Class 2. Class 1 Despite their incredible abundance, Class...components present in the Cascade complex. Figure 2: Schematic of a Cascade complex (blue subunits) with...

  18. Plasmids 101: How to Verify Your Plasmid Using a Restriction Digest Analysis

    Type
    Blog Post
    Published
    Aug. 28, 2014, 3:34 p.m.
    ...Diagnostic restriction digests are comprised of 2 separate steps: 1) incubating your DNA with restriction...which cleave the DNA molecules at specific sites and 2) running the reaction on an agarose gel to determine...including a 1.2 kb insert. The plasmid was digested with 2 unique enzymes (HindIII and BamHI) and run on an agarose...to serve as a reference band. The uncut DNA (lane 2) shows 3 possible plasmid conformations, with relaxed...will allow you to approximate the DNA mass of comparably intense samples of similar size. Run the gel ...gel at 80-150V until you have good separation between your bands. Stopping the gel when the bromophenol ...need to run the gel for longer to achieve good separation of larger DNA fragments. I hope these tips ...

  19. Screening for Successful Genome Editing with Digital PCR

    Type
    Blog Post
    Published
    March 30, 2017, 2:30 p.m.
    ...resulting from non-homologous end joining (NHEJ) (2-5), depending on your desired genome edit. Since exploitation...each sample can be quickly calculated (details in [2]) and can be used to quantify rates of mutation in... have been reported for mismatch nuclease assays (2, 6, 7). Ability to distinguish between mono-allelic...assays, in addition to testing their performance (2). Remember that although digital PCR is a great screening...PMID: 24509632. PubMed Central PMCID: PMC4063274. 2. Findlay, S. D., Vincent, K. M., Berman, J. R. & Postovit...96-well plate without prior quantification. Comparatively, mismatch assays require between 200 and 500... in a high background of wild type targets. Comparatively, detection limits of about 5% mutant targets...

  20. Finding nucleic acids with SHERLOCK and DETECTR

    Type
    Blog Post
    Published
    April 16, 2020, 1 p.m.
    ... C-Y, Guevara H, Wadford DA, Chen JS, Chiu CY (2020) CRISPR–Cas12-based detection of SARS-CoV-2. Nature... (2015) Discovery and Functional Characterization of Diverse Class 2 CRISPR-Cas Systems. Molecular Cell... can be used to diagnose the presence of SARS-CoV-2 RNA, but inadequate access to reagents and equipment...named this system SHERLOCKv2 for SHERLOCK version 2. Here are some of the improvements: SHERLOCKv2 uses... a protocol for using SHERLOCK to detect SARS-CoV-2 RNA. The test is started using RNA purified from patient...recently adapted the DETECTR platform to detect SARS-CoV-2 using a lateral flow strip format. They published ...Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System. Cell 163:759–771 . https://doi....
Showing: 21 - 40 of 393 results