Skip to main content
Addgene
Open Menu

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

Information

In Category

We narrowed to 303 results for: hal.2

Showing: 121 - 160 of 303 results

  1. Treating Muscular Dystrophy with CRISPR Gene Editing

    Type
    Blog Post
    ...nonsense-mediated decay. Although Nelson et al. observed only 2% genome editing in one experiment, they found the ... cells throughout the body, especially stem cells 2. Remove any immunogenicity of the AAV vector Once ...Science. 2015 Dec 31. doi:10.1126/science.aae0169 2. Long C, Amoasii L, Mireault AA, McAnally JR, Li H...Precision genome editing faces the challenges above and more. One chief challenge is upregulating HDR in mature...complex oligonucleotide treatment comes with many challenges, researchers have begun to explore genome editing... the time to clinical approval. Even with the challenges precision editing faces, it’s encouraging to ...WX, Scott DA, Gootenberg JS, Kriz AJ, Zetsche B, Shalem O, Wu X, Makarova KS, Koonin EV, Sharp PA, Zhang...

  2. CRISPR 101: Epigenetics and Editing the Epigenome

    Type
    Blog Post
    ...form or as ready-to-use lentivirus. Figure 2: dCas9-Tet1 demethylates cytosines at promoters and...pX330a dCas9-LSD1 for targeted removal of H3K4me1/2 and H3K9me2 histone modifications. Like the TALE-LSD1...approach for targeted DNA demethylation. Cell Discovery 2: . https://doi.org/10.1038/celldisc.2016.9 Find plasmids... demethylase to demethylate enhancer regions (Mendenhall et al., 2013). By comparing gene activation when...sorting and selection of transduced cells. Grant Challen’s lab also created constitutive (pCMV-dCas9-D3A)...contributed to updating this article. References Haldeman JM, Conway AE, Arlotto ME, et al (2018) Creation...Fowler T, Rees R, Kramer A, Martens A, Edwards JR, Challen GA (2016) Reprogrammable CRISPR/Cas9-based system...

  3. CRISPR Methods for Bacteria: Genome Engineering, CRISPRa, CRISPRi, Base Editing, and More

    Type
    Blog Post
    ... pCRISPomyces-2 contains Cas9 and a gRNA cassette. The simpler system of pCRISPomyces-2 displays a higher...and a subsequent curing protocol that requires only 2-3 hours incubation. E. coli and T. citrea scarless...four major components including (1) a CRISPR RNA, (2) four proteins forming the QCascade DNA-targeting ...DSB) are lethal. Addgene depositor Luciano Marraffini’s lab took advantage of this lethality to design...organisms, CRISPR-induced double stranded breaks are lethal because the non-homologous end-joining (NHEJ) repair...Cas9 will cleave the gene of interest, inducing a lethal DSB.     This system is distinct from those used...While the activation of transcription is a more challenging problem than repression, scientists have deposited...

  4. Antibodies 101: Reading a Flow Plot

    Type
    Blog Post
    ...A-B+ cells. If you look at the dot plot in Figure 2, you might assume that all cell populations are positive... the different populations. In the case of Figure 2, one population is B- and another two are B+. Our ...are in the lower right corner.      Figure 2: This flow plot has two populations that are B+ and...from flow cytometry experiments can be a little challenging to understand if you’ve never done any flow. ...., Sait, K., Anfinan, N., Al-Wasiyah,  mohammad khalid, Huwait, E., Gari, M., & Al-Qahtani, M. (2018)....

  5. Quickest Way to Deposit Plasmids: The Deposit Spreadsheet

    Type
    Blog Post
    ...our deposit spreadsheet to complete Step 1. Steps 2 and 3 are usually very easy — our tech transfer team...columns are shown in red in the spreadsheet (Figure 2) and are described in Table 1. Some cells have drop-down...Filling out and sending the deposit spreadsheet Figure 2: Example of the deposit spreadsheet showing the first... (budding yeast), S. pombe (fission yeast), A. thaliana (mustard weed), O. cuniculus (rabbit), synthetic...

  6. Pathways Over Time Plasmids Engage Students in Functional Genomics Research

    Type
    Blog Post
    ... undergraduate students (1, 2), but educators face significant challenges in designing suitable projects... by western blotting in other experiments (Figure 2).   Complementation reveals conservation of function...Biology Education: A Call to Action, Washington, DC. 2. President's Council of Advisors on Science and Technology...

  8. Three Key Considerations for Precise Neuronal Targeting Using AAV Technologies

    Type
    Blog Post
    ... characteristics of select AAV serotypes in Table 2 from the paper. Delivery of AAV into the brain can...routes for neuroscience see Table 1 from the paper. 2. Manipulate your cell of interest: Cell-type specific...development, learning, and behavior poses many challenges in neuroscience with many brain regions, neuronal...

  9. AAV Titration by qPCR Using SYBR Green Technology

    Type
    Protocol
    ... 10 90 2 x 10 8 10 of 2 x 10 8 dilution 90 2 x 10 7 10 of 2 x 10 7 dilution 90 2 x 10 6 10 of 2 x 10 6...6 dilution 90 2 x 10 5 10 of 2 x 10 5 dilution 90 2 x 10 4 10 of 2 x 10 4 dilution 90 2 x 10 3 Pro-Tip...molecules/μL To obtain a solution at 2 x 10 9 molecules/μL: 1.59 x 10 11 / 2 x 10 9 = 79.8X dilution ...your standard curve plasmid (2 x 10 9 stock made in step #1): Volume of 2 x 10 9 stock or previous dilution...stock 45 uL 10X 10X Dilution 2 5 uL Dil. 1 95 uL 20X 200X Dilution 3 20 uL Dil. 2 80 uL 5X 1000X Dilution ...time required: ~3 h Workflow Timeline Plate set-up: 2 h qPCR run: 1.5 h Data analysis: 30 min Equipment ...single channel pipette 1–10 µL multichannel pipette 2–50 µL multichannel pipette 20–200 µL multichannel ...

  10. Plasmids 101: Degron Tags

    Type
    Blog Post
    ...required components are 1) a SMASh-tagged protein and 2) asunaprevir (small molecule). dTAG The degradation...gone through an additional round of evolution (AID-2) to reduce some of the leakiness associated with the...Adapted from Nabet et al., (2018).   HaloTag The HaloTag was initially developed by Promega as an ...the Halo-tagged protein in question, it is targeted to the proteasome for degradation. The HaloTag is ...straightforward system — HaloTag and its PROTAC are all that is needed to facilitate degradation. HaloTag is also the...visualization of proteins of interest. A fluorescent Halo ligand can be added to media, buffer, etc., which...proteolysis-targeting chimera (PROTAC) was developed for HaloTag. PROTACs are engineered molecules which target ...

  11. Savvy Advocates Needed to Navigate a Scientific Enterprise in Flux

    Type
    Blog Post
    ...is funded have been well documented over the past 2 years (Alberts et al, PNAS 2014; Taylor, eLife, 2016...PMID: 24733905. Pubmed Central PMCID: PMC4000813. 2. Taylor, Justin Q., et al. “Avoiding a lost generation...since the Future of Research Symposia Ongoing challenges facing the scientific enterprise and the way ...landscape of the scientific enterprise will provide challenges and opportunities for us all.  For example, implementation...categorize all postdocs at institutions, will be challenging. At the same time, there is much opportunity ...the pressure on for further increases will be challenging, but will be aided by the development and implementation... is an important way to push the balance from challenge to opportunity. We hope to see you at the upcoming...

  12. Cellosaurus: A Cell Line Information Database

    Type
    Blog Post
    ...groups, such as their vaccine production, SARS-CoV-2 research, or adenovirus packaging cell lines dataset...-Line Knowledge Resource. J Biomol Tech. 2018; 29(2): 25-38....split you got from the graduate student down the hall who got it from some technician downstairs who got...

  13. CRISPR 101: Anti-CRISPR Proteins Switch Off CRISPR-Cas Systems

    Type
    Blog Post
    ...base pair to another base pair at a specific site. 2. Temporal, spatial, or conditional control of CRISPR...difficult-to-engineer Sulfolobus islandicus rod-shaped virus 2 (Mayo-Muñoz et al., 2018). Only viral particles that...Editing of Sulfolobus islandicus Rod-Shaped Virus 2. Viruses 10:695 . https://doi.org/10.3390/v10120695...that have the gene deletion could replicate when challenged with the S. islandicus CRISPR-Cas system. 5. ...

  14. Antibodies 101: Flow Compensation

    Type
    Blog Post
    .... Let's look at an experimental example in Figure 2. Figure 2A shows the sample prior to compensation.... line with the other populations.   Figure 2: Compensation example based on experimental sample..., you will see the populations move, as in Figure 2, following compensation. Figure 3: Flow plot...Compensating between your fluorescent markers can be challenging! But we hope this guide can help you get started...

  15. Antibodies 101: Single Chain Fragment Variables (scFvs)

    Type
    Blog Post
    ...panning and scFv characterization.    Figure 2: Phage display starts with a pooled library of scFvs...the short epitope GCN4, also called a scaffold; and 2) a GCN4 binding scFv that’s fused to GFP. When the...Fv (Variable Domain) (scFv) Fragments. Antibodies 2:193–208 . https://doi.org/10.3390/antib2020193  Khantasup... the body or self antigens, and toxins that are lethal to animals.  In the clinic, scFvs’ size also provides...plasmid in bacteria (Ahmed et al., 2012). One challenge of bacterial expression is the proper formation...

  16. Top Requested Lentivirus and AAV of 2016

    Type
    Blog Post
    ... tropism and axonal transport of AAV serotypes 1, 2, 5, 6, 8 and 9 in the mouse brain. PLoS One. 2013 ...PMID: 24086725. PubMed Central PMCID: PMC3785459. 2. Krashes MJ, Koda S, Ye C, Rogan SC, Adams AC, Cusher...makes generation of high titer Cas9 lentiviruses challenging. This cytotoxicity also causes growth rate variability...):337-47. PubMed PMID: 12595892. 4. Sanjana NE, Shalem O, Zhang F. Improved vectors and genome-wide libraries...

  17. Hot Plasmids: Summer 2025

    Type
    Blog Post
    ...RNA guide (known as an omega RNA, or ωRNA) (Figure 2), but TnpB is small enough to be carried by viral ...than a third of the size of Cas9). Figure 2: Comparison of TnpB (A) and Cas9 (B) genome editors...Figure 5: Mechanism of ATLASCre. VAMP2 = synaptobrevin 2; At = ALFA-tag; BACE = β-secretase; AF = AMPA.FingR...perform neuronal tracing, anterograde tracing poses challenges, from non-selective anterograde transport to ...

  18. Hot Plasmids: Winter 2025

    Type
    Blog Post
    ...resistance to the herbicide chlorsulfuron (Homo line 2) or wild type (WT), two weeks after herbicide was ... CLIPf tag and a C-terminal SNAPf tag; see Figure 2), the authors developed a cost-effective expression... when associated with microtubules. Figure 2: CLIP-SNAP fusion proteins for single-molecule FRET... developed in human cells, has been uniquely challenging to apply in dicotyledonous plants, or dicots....

  19. The AAVantages of AAV in CRISPR Screens

    Type
    Blog Post
    ...handled at BSL-1, while lentivirus requires a BSL-2 setup. This requires a less involved setup when working...infection rates in your experiment. Figure 2: Visual representation of common AAV serotypes used..., 10(1). https://doi.org/10.1038/s41467-019-12449-2 Pupo, A., Fernández, A., Low, S. H., François, A.,.... V., Teyssier, N., Pan, A., Danner-Bocks, S., Sanghal, T., Yadanar, L., Tian, R., Ma, K., Palop, J. J...

  20. Production of Virus in Insect Versus Mammalian Cells

    Type
    Blog Post
    ...) 38 kb Cells HEK-293T Sf9 Time to collection 2–4 days 5–10 days Purification Ultracentrifugation...Centrifugation Titration time ~1 day (AAV); ~1 week (lenti) 2–10 days Initial titers Higher Lower Resources... a factory to produce adeno-associated virus type 2 vectors. Human Gene Therapy, 13(16), 1935–43. https...Come From and What Do They Mean? Overcoming the Challenges of Lentivirus Production Tips for Titering Your...

  21. Tips for arabidopsis transformation

    Type
    Blog Post
    ...Arabidopsis thaliana seedlings following floral dip transformation’, Plant Methods. BioMed Central, 2(1), p....PMID: 24032037. PubMed Central PMCID: PMC3759027. 2 Harrison, S. J. et al. (2006) ‘A rapid and robust ...p. 19. doi: 10.1186/1746-4811-2-19. PubMed PMID: 17087829. PubMed Central PMCID: PMC1636043. 3. Kyndt,...

  22. AAV Production in HEK293 Cells

    Type
    Protocol
    ...430825, 175 cm 2 Cellstack 5, Corning 3319, 3180 cm 2 Cellstack 2, Corning 3269, 1272 cm 2 Heat-inactivated...: 50 mM Tris HCl, 150 mM NaCl, 2 mM MgCl 2 Add the following to the 2 L sterile bottle: 1836 mL deionized... solution can be stored at 4 °C for up to 2 months. After 2 months, discard the tube and thaw a new working... mL of PBS. Aspirate PBS and add 2 mL of 0.05% Trypsin/EDTA. Wait ~2 min. Neutralize trypsin by adding...Cell-Stack (CS5) (Link opens in a new window) (3,180 cm 2 - the same surface area as 21 x T-175 flasks). Cell...flasks. Workflow Timeline Day 0: Seed cells in CS2 Day 2: Seed cells in CS5 Day 3 (am): Transfect cells Day...Biological Safety Cabinet 0.5–10 µL single channel pipette 2–20 µL single channel pipette 20–200 µL single channel...

  23. Summer SciComm: Science Storytelling

    Type
    Blog Post
    ... (1) the struggle between two opposing forces or (2) the result of competing desires or (3) the presence... an introduction, and a resolution.   Fig. 2: The structure of a story - not just for fiction! ... and what can be left out, which can be a big challenge!  The resolution Throughout your conflict, you...

  24. PiggyBac-ing Through the Genome Editing Field

    Type
    Blog Post
    ...without leaving behind any accessory DNA sequences [2], [3]. Simply put, the piggyBac™ transposon containing...with a Footprint-Free™ genome edit.   Figure 2: Editing DNA using the piggyBac™ transposon selection...PMID: 26211958. PubMed Central PMCID: PMC4663986. 2.  A. M. Singh, V. V Adjan Steffey, T. Yeshi, and D...CRISPR/Cas9, TALENs, and ZFNs). However, several challenges still remain, such as making gene edits completely...nuclease technology with the piggyBac™ system, these challenges can be considerably reduced. The piggyBac™ transposon...

  25. 28 Hot Plasmid Technologies from 2015

    Type
    Blog Post
    ... Zetsche et al., Nat Biotechnol. 2015 Feb 2;33(2):139-42. doi: 10.1038/nbt.3149. Light-inducible ...and on your favourite signalling pathways (Figure 2). They are all available now at Addgene. 1 Lungu...20;19(4):507-17. 10.1016/j.chembiol.2012.02.006. 2 Guntas et al., Proc Natl Acad Sci USA 2015 Jan 6; ...Nihongaki et al., Chemistry and Biology. 2015 Feb 19; 22(2):169-74. High efficiency cloning for enzyme screening...inducible, gene knockdown with shRNA or shRNA-miR30, (2) tamoxifen-inducible CreERT2 gene deletion, (3) simultaneous...different fluorescent proteins, 8 constitutive promoters, 2 includible promoters, 3 polyA terminators and various...of plasmids required for each input/output system; 2) removing all chemically inducible promoters; 3) improving...

  26. Choosing the B(right)est Fluorescent Protein: Photostability

    Type
    Blog Post
    ...implies that reducing the power 2-fold does not reduce photobleaching by half. How exactly the photobleaching...prolonged time). Other illumination strategies, such as 2-photon-excitation, selective plane illumination, TIRF...described by the t½, which is the time after which half of the initial fluorescence intensity remains. The...

  27. INTEGRATE: Bacterial Genome Engineering Using CRISPR-Transposons

    Type
    Blog Post
    ...TniQ for targeting (Halpin-Healy et al., 2020). Genomic targets require an upstream 2-bp PAM sequence for...cells of the plasmid after integration. Figure 2: (A) Streamlined VchINT expression constructs. (B)...proteins to form the Cascade targeting complex is a hallmark of Type I CRISPR-Cas systems, in contrast to single-protein...creates a 5-bp target-site duplication (TSD), a hallmark characteristic of the Tn7-family of transposons...follow him on twitter @Vopinator.     References: Halpin-Healy TS, Klompe SE, Sternberg SH, Fernández IS...doi.org/10.1038/s41586-019-1849-0 Klompe SE, Vo PLH, Halpin-Healy TS, Sternberg SH (2019) Transposon-encoded...

  29. History of CRISPR Cas - A tale of survival and evolution

    Type
    Blog Post
    ...phages but to also destroy the phage upon re-exposure (2, 3). More specifically, specialized Cas proteins snip..., accessed 25 November 2013. Author: James Atmos (2). Many CRISPR/Cas moieties carry out these functions...defense against invading phages. Rising to the challenge - Evolution of anti-CRISPR genes in phages The...

  30. The PAM Requirement and Expanding CRISPR Beyond SpCas9

    Type
    Blog Post
    ...is unique compared to the rest of the genome and (2) the sequence is located just upstream of a Protospacer...pyogenes Cas9 variants with varying PAM sequences, 2) the use of Cas9 homologs derived from species other...Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System. Cell 163:759–771 . https://doi....WX, Scott DA, Gootenberg JS, Kriz AJ, Zetsche B, Shalem O, Wu X, Makarova KS, Koonin EV, Sharp PA, Zhang...

  31. Twenty Years of Sharing: Expanding Accessibility

    Type
    Blog Post
    ...China, Korea, Japan, Brazil, India, and Mexico. Table 2: Addgene distributors Distributor Partnership...distribution after we partnered with them (Figure 2). In others, the distributor helped solve an issue...temperature for the duration of their journey. Figure 2: Annual distribution of items to countries with a ...persisted despite our best efforts. With a little over half of our orders coming from researchers outside the...

  32. Hot Plasmids: Fall 2024

    Type
    Blog Post
    ...improve solubility and stability (Figure 2).      Figure 2: Crystal structures of CRBNmidi alone...fluorescent tags in Caenorhabditis elegans. Genetics, 228(2), iyae126. https://doi.org/10.1093/genetics/iyae126... Zhong, B. L., Elliot, J. M., Wang, P., Li, H., Hall, R. N., Wang, B., Prakash, M., & Dunn, A. R. (2024...

  33. Tips for CRISPR Gene Editing in Mice

    Type
    Blog Post
    ... than control, 3; same as control (Cetn1/sgRNA1), 2; darker than control, 1; very dark). Brighter fluorescence...you can hold off on genotyping until the pups are 2-4 weeks old and use the ear tag method (creating different...PMID: 24253446. PubMed Central PMCID: PMC3875854. 2. Mashiko, Daisuke, et al. "Generation of mutant mice...stranded break at the target site, allowing the two halves of EGFP to recombine by homology directed repair...

  34. Filming Science Videos in the Age of Social Distancing

    Type
    Blog Post
    ... Figure 2: Ironing boards. Good as a WFH desk, and as a video control center.   Cycle 2: Zooming ...ironing board, giving it the perfect height (Fig. 2). We recorded video using a simple, external video...small operation, so sometimes it can be a big challenge. But ask any filmmaker or videographer and they...

  35. With an Eye Towards the Future, We Look Back at the March for Science

    Type
    Blog Post
    ...scientists have 1) the courage to make mistakes and 2) the integrity to recognize these mistakes quickly...She studies beta-cell function and failure in Type 2 Diabetes. Birmingham, AL, USA 33.5207°N 86.8025°W ...available to people who want to join the dialog and 2) remember that science should not be partisan: everyone...the University of California Berkeley. It’s been half a year since the march for science on April 22, ...present at the lab bench," Liu, a UM undergraduate, challenged us. I worried the march would be a simple, unquestioning... fingers of skepticism to defend an agenda that halts current data collection, destroys existing data,...

  36. Fluorescent Proteins 101: Monitoring Cell Mobility Using Fluorescent Proteins

    Type
    Blog Post
    ... fluorescent proteins expressed in the specimen.  2. Fluorescence Activated Cell Sorter (FACs) A machine...photostability of your fluorescent protein when using 2-photon microscopy as this hasn't been well reported...PMID: 18703742. PubMed Central PMCID: PMC2606057. 2. Chtanova, Tatyana, et al. "Dynamics of neutrophil... lymph nodes at different times after antigen challenge, scientists were able to track where GFP labelled...et al. 2016, Cavanagh et al. 2008)? Technical challenges of two photon microscopy Intravital imaging is... at steady state or under physio-pathological challenge. However technical specificities have to be taken...

  37. Plasmids 101: Terminators and PolyA signals

    Type
    Blog Post
    ...due to the presence of additional helper sequences (2-3). As alluded to above, termination and polyadenylation...and translation processes as well.   Figure 2: The conserved eukaryotic polyadenylation signal directs...processing and contributes to variability in RNA half-life, and ultimately gene-expression. Polyadenylation...39322032   No terminator is 100% efficient at halting transcription of the template and initiating the...

  38. Illuminating Epigenetics with A FRET Based Biosensor

    Type
    Blog Post
    ...FRET signal back again. The Ting lab constructed 2 versions of this reporter to target the K9 (pcDNA3... 525.7570 (2015): 533-537. PubMed PMID: 26352475. 2. Bygren, Lars O., et al. "Change in paternal grandmothers...real-time, posing a huge challenge to studying epigenetic changes over time.  This challenge was difficult to ...

  39. Selecting Your Plasmid Purification Kit

    Type
    Blog Post
    ... environmental sustainability in mind (Table 2). Table 2: Sustainability features highlighted in plasmid...debris”; and then clean-up the plasmid DNA (Figure 2). For most kits, the steps for lysing cells and removing...together — no mixing and matching!   Figure 2: Plasmid isolation workflows are similar across kits...inks Reduced cardboard packaging by more than half 70% recycled materials Sustainable forestry ...

  40. Visualizing Genomic Loci with CRISPR-Sirius

    Type
    Blog Post
    ...that the signal to noise ratio can be increased from 2-fold (by CRISPRainbow) to 3-fold with CRISPR-Sirius...CNV) analysis of the U2OS cells.   Figure 2: CRISPR-Sirius tricolor labeling. (A) Diagram of sgRNA-Sirius...guide sgRNA-Sirius-8XMS2 which will recruit the HaloTag with red fluorescent ligands to the loci of interest...
Showing: 121 - 160 of 303 results