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We narrowed to 281 results for: hal.1

Showing: 1 - 20 of 281 results

  1. CRISPR Challenges: Standardization and Homology Directed Repair

    Type
    Blog Post
    Published
    Feb. 14, 2018, 2:36 p.m.
    ...with CRISPR has been challenging, with protocols using dsDNA templates achieving ~1-10% efficiency. Easi-CRISPR...B. Gurumurthy, and Masato Ohtsuka. Nat Protoc. 13(1) (2018):195-215. PubMed PMID: 29266098 Creating knock-in...that this basic concept of gRNA design is still challenging for researchers using CRISPR, and that standardizing...paraphrased in the tweet below. The complexity and challenges of homology directed repair (HDR) are echoed ...s a list of blog posts to help you tackle the challenges of DNA repair (with bonus gRNA design resources...

  2. Designing Your Chalk Talk for the Academic Job Interview

    Type
    Blog Post
    Published
    March 12, 2019, 1:37 p.m.
    ...acceptable. Aims should be mechanistic, doable by 1-3 people in 1-5 years, and contribute to resolving the overarching...possible, do at least two full length practices (~1 h) and get feedback. It’s also helpful to practice...I include in my chalk talk?" So what exactly is a chalk talk? On the surface, a chalk talk is quite simple...present a "chalk talk." However, few, if any, faculty job candidates have seen an actual chalk talk. Their...first exposure to a chalk talk is usually their own. This is a problem. The chalk talk is effectively ... hone a chalk talk. Yet, I often hear, "I have an interview next week and it includes a chalk talk. What...steps to chalk talk preparation: Define your research vision. Organize your vision into a chalk talk format...

  3. Countdown to Halloween @Addgene

    Type
    Blog Post
    Published
    Oct. 29, 2014, 5:26 p.m.
    ...glorious trophy. Sure it’s plastic and you have to share 1 trophy among 6 people, but you’ve earned bragging ...Halloween is only 2 days away and here at Addgene we couldn’t be more excited! As a small, tight-knit...things: fun, teamwork, and competition. And our Halloween celebration this Friday will combine all of these...these. Addgenies have been organizing Halloween festivities for years – searching our photo archives I...destroyed to protect the innocent revelers. Our Halloween costume contests have become an annual tradition...colleagues. Success is sweet – as sweet as all that Halloween candy you’ll gorge yourself on in the coming days...Hopefully, I’ve made clear how excited we get for Halloween and how seriously we take costume design. In case...

  4. Streaking for Single Colonies: The Streak Plate Challenge

    Type
    Blog Post
    Published
    March 2, 2021, 2:15 p.m.
    ...technique. Time for the #BioSci3319 #StreakPlateChallenge part 1. Lab B needs a redo :-( @SandTBioSci ... the lab used three different species for the challenge, but one species always outcompeted the other ...it would be a perfect fit for the streak plate challenge. Chromoproteins are a subset of the fluorescent... distinguish colonies from one another in the challenge. To create a successful streak plate, first the...colonies in the last quadrant after incubation. The challenge is usually successful for the students. “They ...colonies. I should probably make it a bit more challenging, potentially by using a thicker bacterial suspension...transformation efficiency.  Aside from the Streak Plate Challenge, Westenberg’s course includes Winogradsky columns...

  5. Six Spooky Science Stories and Halloween at Addgene

    Type
    Blog Post
    Published
    Oct. 31, 2019, 1:06 p.m.
    ...Molecular and cellular endocrinology 215.1-2 (2004): 1-10. PubMed PMID: 15026169. Soo, Rochelle M., et al...another snail, completing its lifecycle. Halloween genes The Halloween genes are a set of cytochrome P450 genes...incorporate science themes into our fun activities and Halloween is no exception. For the past 10+ years, Addgenies...ping pong table. This year, in addition to the Halloween contest, we’ve also carved pumpkins (yes, Blugene...melanogaster (Gilbert, 2004). Mutations in these genes are lethal, associated with defective exoskeleton formations...1983.3 (1983): 813-816. Gilbert, Lawrence I. "Halloween genes encode P450 enzymes that mediate steroid...company culture at Addgene See some of our previous Halloween costume contests ...

  6. 3 Challenges in Plant Synthetic Biology

    Type
    Blog Post
    Published
    July 22, 2014, 5:46 p.m.
    ... had to navigate the challenges involved in plant synthetic biology. Challenge #1: Public perception of...facilitates plant science. Challenge #3: Intellectual property There is another challenge associated with molecular... of well-characterized genetic tools make it challenging to engineer a specific function in these multicellular...fibers. And because working with plants can be challenging, there are a lot of unexplored areas in plant...manner. At Revolution Bio, we are addressing this challenge directly by developing a plant biotechnology product...inspire and open a new conversation about GMOs.  Challenge #2: Technical obstacles to plant synthetic biology... a product without the use of these parts is challenging, and it’s made even more difficult by the fact...

  7. Overcoming the Challenges of Lentiviral Production

    Type
    Blog Post
    Published
    Aug. 7, 2018, 12:31 p.m.
    ...is best for larger genes such as Cas9. See figure 1 for an example of how changing transfection ratios...tools, producing lentivirus, can pose certain challenges. Whether choosing a system that is the best fit...will provide an overview of some of the common challenges associated with producing and using lentivirus...make these optimization experiments a bit more challenging. With these considerations in mind, when planning...

  8. The Challenges of Cell Culture

    Type
    Blog Post
    Published
    May 5, 2016, 2:30 p.m.
    ...testing. You can find Nick on LinkedIn. References 1. Skloot, Rebecca, and Bahni Turpin. The immortal life...

  9. Targeting HIV-1 with CRISPR: Shock and Kill or Cut it Out?

    Type
    Blog Post
    Published
    May 10, 2016, 2:30 p.m.
    ...plasma HIV-1, improving life expectancy and quality for HIV-1 patients. However, ART is not an HIV-1 cure. ...activated HIV-1, measured via HIV-1 promoter-driven GFP expression. In cell lines that can produce HIV-1 toxic...with HIV-1. For two HIV-1 strains, Cas9/gRNA lentiviral expression significantly reduced the HIV-1 copy number...currently infected with the lentivirus HIV-1. Today, HIV-1 can be controlled with antiviral therapies ...HIV-1 transcription and destroy infected cells, the other using wild-type Cas9 to remove the HIV-1 genome...take on HIV-1 in vitro, and what obstacles must be overcome for clinical success. ART and HIV-1 reservoirs...Cutting HIV-1 out of the genome with CRISPR/Cas9 Before the introduction of CRISPR, HIV-1 genome editing...

  10. Live and Let Dye: Self-Labeling Protein Tags

    Type
    Blog Post
    Published
    April 16, 2024, 1:15 p.m.
    ...intracellular targets. Nature Communications, 7(1), Article 1. https://doi.org/10.1038/ncomms10778 Erdmann...molecules in vivo. Nature Biotechnology, 21(1), Article 1. https://doi.org/10.1038/nbt765 Lavis, L. (2016... envelope design. Nature Communications, 14(1), Article 1. https://doi.org/10.1038/s41467-023-40788-8 ... capping protein. Nature Communications, 14(1), Article 1. https://doi.org/10.1038/s41467-023-39655-3 ...up linked to whatever molecule you choose (Figure 1). Just express the fusion protein and add a specific...proteins, opening up new applications.   Figure 1: Live-cell labeling strategies. The green structure...minimally-disruptive genetic tag on the protein of interest. Table 1: Summary of popular self-labeling tag systems ...

  11. Split Fluorescent Proteins for Studying Protein-Protein Interactions

    Type
    Blog Post
    Published
    May 3, 2022, 1:15 p.m.
    ...General Design Figure 1: Two proteins are fused to the FP(11) and FP(1-10) fragments. When Protein...and 11 to yield large (1-10) and small (11) fragments. In several cases an FP(1-10) fragment may be compatible...Description Article PI Blue EBFP2 EBFP2(1-10) and Capri(1-10) for use with GFP(11) Multiplexed labeling...Commun. 2017 Bo Huang mNeonGreen3 mNG3K(1-10) and mNG3A(1-10) for use with mNG2(11) Improved yellow-...sfCherry2 sfCherry2(1-10) and sfCherry2(11), also photo-activatable variant PAsfCherry2(1-10) Improved split...brings the pieces of the FP back together (Figure 1). This method is generally known as Bimolecular Fluorescence...even from a different base FP (for example, EBFP2(1-10) can be used with GFP(11)). Some of the articles...

  12. Antibody Validation for Flow Cytometry

    Type
    Blog Post
    Published
    Sept. 10, 2024, 1:15 p.m.
    ...dye, respectively. WT and KO cells were mixed in a 1:1 ratio and fixed in 4% PFA and permeabilized in 0.1%...antibodies were diluted to 1 µg/mL except for 14558, which was used at 0.35 µg/mL (1/100). * = monoclonal antibody...through their HLDA workshops. Let’s use TIM-1 (Single pass type-1 membrane protein), which has been designated...specific antibody:target combination of interest (Figure 1). It can therefore be helpful to try all three when...to label an intracellular target.   Figure 1: HCT 116 WT and SYT1 KO cells were labelled with a...cells were stained with the indicated Synaptotagmin-1 antibodies and corresponding Multi-rAb CoraLite® Plus...between the wild-type and the KO population (Figure 1), further optimization can be performed to both maximize...

  13. Lentiviral Vector Uses and Overview

    Type
    Blog Post
    Published
    May 19, 2016, 2:30 p.m.
    ...HIV-1 were transduced with a lentiviral vector containing an antisense sequence against the HIV-1 envelope...Trono and collaborators turned to the lentivirus HIV-1, well-known to infect non-dividing cells. The first...plasmids. The packaging plasmid carried a mutated HIV-1 provirus that couldn’t package itself due to a few...plasmid encoded the desired transgene flanked by HIV-1 long terminal repeats (LTRs) that facilitate viral... targeting and tracing cells in the brain (Figure 1). 2nd generation lentiviral packaging system The graphic...infectivity, is often used as a substitute for HIV-1 Env, which can only infect CD4+ cells. 3rd generation...lentivirus in the comments section below! References 1. In vivo gene delivery and stable transduction of ...

  14. AAVs CREATed for Gene Delivery to the CNS and PNS

    Type
    Blog Post
    Published
    Sept. 28, 2017, 2:01 p.m.
    ...nervous system (CNS). AAVs are popular tools because: 1) their genomes are easy to manipulate, 2) they have... stained for a neuronal marker). Check out tables 1 and 2 below for a summary of the quantified transduction... AAV vectors. What can these AAV’s be used for?  1. Multi-color labeling of neurons Multi-color labeling... dendrites extend to. The keys to this method are 1) high levels of color diversity so that cells are ...AAV.PHP.eB 69 +/- 4% 55 +/- 7% 76 +/- 5% Table 1: Transduction efficiency of AAV-PHP.B versus AAV-PHP.eB...expression into a two part system (figure 4d):  Part 1: A cocktail of three AAV vectors expressing RGB proteins...neuronal transduction efficiency, as shown in tables 1 and 2, with a cell-type specific promoter or enhancer...

  15. X-CHIME: Context Dependent Germline Knockout in Immune Cells

    Type
    Blog Post
    Published
    Jan. 16, 2024, 6:13 p.m.
    ...in the immune system. Nature Immunology, 25(1), Article 1. https://doi.org/10.1038/s41590-023-01689-6 ...developed immune system was lethal (LaFleur et al., 2024).   Figure 1: Schematic showing the X-CHIME... strains and lentiviral expression vectors (Table 1). The four systems Name Function Plasmid ... GFP (vex) Rosa26-FlpO-ERT2; H11-Cas9 Table 1: X-CHIME systems Proof of Concept These systems were...genes in the immune system. Nature Communications, 10(1), 1668. https://doi.org/10.1038/s41467-019-09656-2... cancer immunotherapy targets but are embryonic lethal, hindering studies post embryogenesis. Using C-...

  16. Tips for a 1st Time CRISPR User (by a 1st Time CRISPR User)

    Type
    Blog Post
    Published
    March 7, 2017, 3:30 p.m.
    ...Pro-Tips: 1.Designing primers against genomic DNA to amplify the gRNA targets was challenging because you...The entire process from start to finish took about 1 month. Cas9 delivery Overview: Before any editing...lentiviral vector, lentiCas9-Blast (52962-LV, Figure 1). Some cells were then used to generate a monoclonal...rest of the cells were frozen down. Figure 1: Cas9 expression in A549 cells transduced with lentiCas9...Western blotting is a loading control. Pro-Tips: 1. It’s all about the MOI. In retrospect I used a way... pool had wildly variable Cas9 expression (Figure 1). This is a problem because Cas9 expression affects...cells were transduced (MOI = 37) and selected with 1 µg/mL blasticidin for 9 days. Single cells from a ...

  17. Harnessing TnpB for Genome Editing: A Compact and Versatile Tool for Your Research

    Type
    Blog Post
    Published
    July 29, 2025, 1:15 p.m.
    ... Oligos Oligo 1: Add 'tcaa' to the 5′ end of your forward guide sequence (see Figure 1). Oligo 2: Add... Figure 1). Perform standard phosphorylation and annealing of the two oligos. Figure 1: Components... promoter for ωRNA (reRNA) expression (see Figure 1). Both plasmids contain a hygromycin resistance gene...provide a step-by-step guide for using a Dra2TnpB. Step 1: Design the 20-nt Guide Sequence Identify a TAM site...protocols (pp. 245–260). https://doi.org/10.1007/978-1-0716-4358-7_20  Karmakar, S., Panda, D., Panda, S... induction of meristems. Nature Biotechnology, 38(1), 84–89. https://doi.org/10.1038/s41587-019-0337-2...1166–1187. https://doi.org/10.1038/s41477-021-00991-1  Qiao, J., Zang, Y., Gao, Q., Liu, S., Zhang, X.,...

  18. Mouse Modeling, Part 2: Breeding and Crossing Mice

    Type
    Blog Post
    Published
    Aug. 6, 2019, 12:55 p.m.
    ...approximately 1 in 16 mice that harbor homozygous alleles at both of your genes of interest. About 1 in 4 mice...In Part 1 of our mouse modeling blog series, we covered techniques that can be used to introduce genetic...mice in your experiment. As you’ve learned in Part 1, there are many types of genetically engineered mice..., and assuming Mendalian patterns of inheritance: 1. Genotype your parental mice In order to ensure that...example of mice that are derived from crosses. In Part 1, we explained how genetic recombination in mice expressing...expressed, or inducible (this is explained more in Part 1 of this series). In any case, Cre is a dominant phenotype...that can impact reproduction include embryonic lethality, a short lifespan, or infertility and can arise...

  19. Degrading DNA with Cascade-Cas3

    Type
    Blog Post
    Published
    Feb. 11, 2025, 2:15 p.m.
    ...Cascade's complexity. Figure 1: Schematic of Class 1 CRISPR types. Class 1 includes type I with a Cas3 ...collection! A “complex” system Cas3 is part of the Class 1 CRISPR family, which means it requires multiple Cas...the brunt of the mechanical work for editing. Class 1 CRISPR systems make up the majority of CRISPR-based...CRISPR-based immune systems in bacteria and archaea. Class 1 is further broken down into different types, with ...making up type I, the most abundant type in the Class 1 category. These type Is can then be further broken...editing in human cells. Nature Communications, 10(1). https://doi.org/10.1038/s41467-019-13226-x Cameron...this complex, developing Cas3 tools has been challenging. It can be difficult to ensure comparable expression...

  20. pCXLE toolkit: Efficient episomal plasmid-based method to reprogram peripheral blood cells to iPSCs

    Type
    Blog Post
    Published
    Dec. 14, 2017, 2:08 p.m.
    ...are based on the Epstein-Barr Nuclear Antigen-1 (oriP-EBNA-1) system where each vector contains a viral ...replication, OriP and the EBNA-1 sequence that encodes for the DNA binding protein EBNA-1 [7]. These two sequences...extra plasmid, pCXWB-EBNA-1 is used in the reprogramming cocktail. pCXWB-EBNA-1 lacks oriP and is unable...expressing additional EBNA-1 transiently. The transiently expressed EBNA-1 increases protein expression...pCXLE vectors listed below in Table 1 are available at Addgene. Table 1: List of pCXLE episomal plasmids,...obtained and the subsequent reprogramming success [1]. Fibroblasts need to be cultured and expanded in ...reprogramming factor cocktail, and viral protein EBNA-1. Methods for reprogramming hiPSCs from PBMCs While...
Showing: 1 - 20 of 281 results