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We narrowed to 853 results for: ELL

Showing: 421 - 450 of 853 results

  1. Plasmids 101: Sequence and Ligation Independent Cloning (SLIC)

    Type
    Blog Post
    Published
    Sept. 26, 2024, 1:15 p.m.
    ...bacteria, so it can be transformed as-is into cells. The cells then get to work fixing up the “damage” and...nicked plasmid together long enough to get it into cells and let them do the repair work. The key is T4 DNA... update, courtesy of Addgene depositor Stephen Elledge. His new method, named sequence- and ligation-independent...assembled in a unique order with no cloning scars. The Elledge Lab showed that a five-piece assembly reaction ...vitro to generate recombinant DNA via SLIC. Li MZ, Elledge SJ. Nat Methods. 2007 Mar;4(3):251-6. Epub 2007...sequence- and ligation-independent cloning. Li MZ, Elledge SJ. Methods Mol Biol. 2012;852:51-9. doi: 10.1007...

  2. Cas13d: Small RNA-targeting CRISPR enzymes for transcriptome engineering

    Type
    Blog Post
    Published
    May 3, 2018, 1:48 p.m.
    ...editor in mammalian cells Not all Cas13d proteins are functional in mammalian cells, but Konermann et al... Ruminococcus flavefaciens (nicknamed CasRx), as well as EsCas13d. Find the Yan et al. plasmids here ...make it possible to target essentially any RNA in a cell. Since catalytically dead dCasRx maintains its RNA-binding...natural diversity of CRISPR systems, only time will tell how large the genome and transcriptome engineering... with RNA-Targeting Type VI-D CRISPR Effectors.” Cell (2018) pii: S0092-8674(18)30207-1. PubMed PMID: ... a WYL-Domain-Containing Accessory Protein.” Mol Cell. (2018) pii: S1097-2765(18)30173-4. PubMed PMID:...

  3. 7 Tips to Secure a STEAM Internship This Summer

    Type
    Blog Post
    Published
    Feb. 2, 2021, 2:15 p.m.
    ... to impress the judges on your ability to tell a compelling narrative that is positive, and forward-looking...size, legibility, and more). Tips on drafting a stellar resume and cover letter can be found on the Addgene...recommenders based on your relationships to the person as well as their current sphere of influence. In other words... for them, the likelier you will end up with a stellar letter of support.        5. Application submission...landscape beyond college. They will also serve you well when you are applying for an entry-level position... as you apply for internships, scholarships, fellowships, and entry-level positions post graduation. ...

  4. A Deep Dive into BioBuilder

    Type
    Blog Post
    Published
    Oct. 11, 2017, 1:44 p.m.
    ...want to learn more about Synthetic Biology. Can you tell me where to start?” is a question more and more ...2011, BioBuilder is the brainchild of Dr. Natalie Kuldell from MIT’s Department of Biological Engineering...from her 20 years of college teaching experience, Kuldell sought to create a program that would capitalize...contribute as part of a team.” As a seasoned educator, Kuldell also knew that the best days in the classroom are...plan or outstanding test scores.  As a result, Kuldell launched the BioBuilder Educational Foundation ...of Professor Richard Murray. Murray’s lab uses cellular extracts to perform transcription and translation...develop and test hypotheses. Students then use cellular extracts as a means to rapidly prototype DNA circuits...

  5. Quick Guide to Working with Drosophila Part 3: Genome Engineering in Flies

    Type
    Blog Post
    Published
    July 28, 2017, 1:30 p.m.
    ...Drosophila cell line, I’ve studied a transcription factor important for T cell differentiation (a cell type ...integration will have eyes that are anywhere from light yellow to red. The variation in color depends on how transcriptionally...vermillion (another gene controlling eye color) and yellow (a gene affecting the body color of the adult fly...regeneration and homeostasis in the Drosophila midgut. Cell. 137, 1343–55 (2009). PubMed PMID: 19563763. PubMed...et al. Digoxin and its derivatives suppress TH17 cell differentiation by antagonizing RORγt activity. ...

  6. Multiplex Genome Editing with CRISPR-Cpf1

    Type
    Blog Post
    Published
    May 9, 2017, 2:12 p.m.
    ...expression and needing to work with a transfectable cell line. 2) They require larger expression vectors... of 4 crRNAs in vitro and when expressed in 293 cells. This allows for a single promoter to drive expression... were used.  With transfection, 6.4% of HEK293T cells had edits at 4 of 4 targets when an array of 4 crRNAs...transfected on a single plasmid. This is versus 2.4% of cells transfected with a pool of plasmids that contained...processing of the crRNA array when expressed in cells, but notice how Zetsche et al reverses the orientation...mouse neurons and in vivo. For these experiments, cells were infected with a 1:1 ratio of two AAVs,  one...RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System. Cell,163(3), 759-771. PubMed PMID: 26422227. PubMed Central...

  7. Back to Bacteria: CRISPR gRNA Multiplexing Using tRNAs

    Type
    Blog Post
    Published
    June 2, 2015, 6:06 p.m.
    ...toolkit in human cells. Nissim L, Perli SD, Fridkin A, Perez-Pinera P, Lu TK. Mol Cell. 2014 May 22;54(...effect of gene knockout in vivo and in vitro, as well as to insert targeted mutations through homologous...advantageous to instead use a preexisting, conserved cellular machinery to process gRNAs. Hijacking tRNA processing...tRNAs are among the most abundant molecules in a cell, this system is clearly also robust enough to ensure...examine the frequency of both indels at each locus, as well as large deletions. They detected fragment deletion...characterized the system in plants, PTGs will likely work well in many different biological systems. Size is also...

  8. 3 Challenges in Plant Synthetic Biology

    Type
    Blog Post
    Published
    July 22, 2014, 5:46 p.m.
    ...timeline from transformation to testing and a lack of well-characterized genetic tools make it challenging ...challenging to engineer a specific function in these multicellular organisms. However, the rewards are great if ...pathways involved in central anthocyanin metabolism are well understood. Additional work has also identified ...tools facilitates plant science. Challenge #3: Intellectual property There is another challenge associated... agricultural companies and carry associated intellectual property concerns – patents and licensing agreements...Biochemistry Department at the University of Manchester as well as the Biology Department at CSU. When not pursuing...enjoys spending time with her husband and two Jack Russell Terriers. More SynBio Reading: Learn more about...

  9. Transferable Skills Guide: Teamwork

    Type
    Blog Post
    Published
    Nov. 28, 2017, 2 p.m.
    ...assemble a crack team of Addgenies that includes: Michelle, the Senior Scientist who coordinates all kit ....” There was a time in human history when an intelligent, motivated person really could know a little ...any favors by trying to go it alone. The help of fellow lab members and collaborators will not only get...will ultimately improve the quality of the work as well. Social interaction is a good thing Part of you...communication Few people actually like meetings, but run well, meetings can be a critical part of any successful...team could have a strong sense of purpose, an excellent leader, and perfectly clear lines of communication...myth. The original Dr. Frankenstein from Mary Shelley’s novel worked alone, but even the movie makers ...

  10. Antibodies 101: Affinity Reagents

    Type
    Blog Post
    Published
    April 14, 2022, 1:15 p.m.
    ...in transfected eukaryotic cells, allowing for them to be used as intracellular inhibitors.  Aptamers are...small enough to be expressed in bacterial and other cell systems. Since the majority of these alternative...the constant region. Nonspecific binding, caused cell receptors for the constant region, is also reduced...binding strength, half-lives, and/or specificity as well. They’re more difficult to source, may not be readily..., peptides, amino acids, drugs, metal ions, and cells. They can be used either with or as fluorophores... may sound. Validated antibodies are still an excellent option for many applications, and frankly, if ...

  11. CRISPR 101: Cas9 vs. The Other Cas(s)

    Type
    Blog Post
    Published
    Dec. 8, 2022, 2:15 p.m.
    ...Deletions in Human Embryonic Stem Cells Using Type I CRISPR-Cas. Molecular Cell, 74(5): 936-950.e5 (2019). 10.1016...a class 2 CRISPR-Cas system. Cell, 163(3): 759-771 (2015). 10.1016/j.cell.2015.09.038 Harrington, B., ...is shared by other Cas enzymes outlined below as well. Cas12a also has significant therapeutic promise... Cas13 are 1). non-specific RNA cleavage and 2). cell toxicity. Cas7-11 does not display collateral RNA...cleavage and is significantly less toxic to mammalian cells than Cas13 (likely due to the lack of collateral...

  12. Human Germline Editing Using CRISPR

    Type
    Blog Post
    Published
    Aug. 10, 2017, 2:19 p.m.
    ... in the embryos, as well as whole exome sequencing of embryo derived-ES cells.They also conducted whole...ssODN repair template in induced pluripotent stem cells made from the patient. With the first gRNA, they...homology-directed repair (HDR) taking place in 41.2% of edited cells. The results were even lower for the second gRNA...13.1% targeting efficiency with only 13% of edited cells employing HDR. Ma et al. moved forward with gRNA...babies.” The differences in editing between iPS cells and embryos make a case for conducting research ...analysis is not possible with IVF, as most of the cells are needed for proper development. Ma et al.’s experiments...

  13. Plasmids 101: Cre-lox

    Type
    Blog Post
    Published
    Jan. 13, 2015, 3:47 p.m.
    ...transgenic animals, embryonic stem cells, and/or tissue-specific cell types: Cre-lox recombination. What.... How can I use Cre-lox? The Cre/lox system is a well-established research tool, especially in the field...downstream of promoters that are active only in certain cell or tissue types, during certain stages of development... recombinase can be expressed only in specified cells or at specified times. Combining this with some ...has been used to successfully introduce Cre into cells of interest. What if I need two separate recombination...2. Site-specific DNA recombination in mammalian cells by the Cre recombinase of bacteriophage P1. Sauer...

  14. Plasmids 101: FLEx Vectors

    Type
    Blog Post
    Published
    April 28, 2016, 2:30 p.m.
    ... in vivo conditional gene rescue experiments, as well as for improvement of optogenetic adeno-associated...delivered using AAV injection, the majority of the cells surrounding the injection site will become infected... ensure that the reporter remains silent until a cell or tissue-specific Cre is provided. Since the FLEx...used to study the physiology or behavior of certain cell populations.  Acknowledgement:Through a partnership...for monitoring Cre-mediated recombination at the cellular level in the mouse. 2003 PubMed PMID: 12665802...FLEX switch targets Channelrhodopsin-2 to multiple cell types for imaging and long-range circuit mapping... Michelle Cronin...

  15. No Llamas Required - Synthetic Nanobodies Against Membrane Proteins

    Type
    Blog Post
    Published
    June 18, 2020, 1:15 p.m.
    ...Single-Domain Camelid Antibodies. Cell 181:1004–1015.e15 . https://doi.org/10.1016/j.cell.2020.04.031 Zimmermann ...coronaviruses and enable the virus to enter host cells. To do this, they injected the spike protein from...camelid antibodies that bind to the spike proteins in cell culture, and inhibit these viruses.  But llama ...hidden in the molecular crevices proteins on the cell surface. Figure 1: Comparison of common antibody...Graham BS, Callewaert N, Schepens B, Saelens X, McLellan JS (2020) Structural Basis for Potent Neutralization...blog Opto-Nanobodies: Using Light to Manipulate Cell Signaling and Protein Purification RANbodies: Reporter...

  16. AAV Vector Quality Control: Going the Extra Mile with NGS

    Type
    Blog Post
    Published
    Sept. 12, 2017, 1:44 p.m.
    ...establishment of in-house sequencing capabilities and Seqwell’s plexWell technology, we can now perform NGS at Addgene... check viral transgene expression in transduced cells whenever possible. This post will describe our workflow...fragments of DNA from the helper plasmids or the cell genome can be packaged inside the vectors during...earlier, it is common to find DNA from the packaging cell genome, bacterial genomes, cloning vectors, and ...future plans How did AAV-68544 become contaminated? Well, we purposely mixed another AAV vector with AAV-...Tasissa M, Kostman M, Tillgren M, Makana Hanley L, Mueller I, Mitsopoulos A, Fan M (2020) A Novel Next-Generation...

  17. Light Sheet Fluorescence Microscopy

    Type
    Blog Post
    Published
    July 16, 2015, 2:30 p.m.
    ...transparent are enabling microscopes to visualize the cellular architecture of whole tissues in 3D with unprecedented..., CST, spinal cord and cranial nerve afferents labelled with a AAV8 expressing eGFP under the human ubiquitin... (2007). Nature Methods, 4, 331–336. Pubmed. P. Keller, M.B. Ahrens.  (2015) Neuron, 85, 462-483. Pubmed... Weber, M. Mickoleit, J. Huisken. (2014) Methods Cell Biol.123, 193-215. Pubmed. W. Spalteholz. (1914)...296, 541-545. Pubmed. A. Ertürk, C.P. Mauch, F. Hellal, F. Förstner, T. Keck,  K. Becker, et al. (2011... Jährlin, C.P. Mauch, C.D. Hojer, J.G. Egen, F. Hellal, F. Bradke, M. Sheng, H.-U. Dodt. (2012). Nature..._the_OpenSPIM_Wiki http://www.janelia.org/lab/keller-lab http://www.physikalischebiologie.de http...

  18. Addgene’s New Way to Help You Share Science

    Type
    Blog Post
    Published
    Nov. 14, 2013, 4:10 p.m.
    ...with a great story to tell? Let us know by emailing us at [email protected] or tell us on Twitter and Facebook...amazing stories to share. I spend a lot of time telling scientists about Addgene’s repository, and explaining...better day at the office but for stronger science as well. This blog is our way of continuing the conversation...whether you’re a graduate student, post-doctoral fellow, lab manager, research technician, or Principal...conversation! Do you have a question about culturing iPS cells? Confused by which system to use for genome editing...

  19. Droplet Digital PCR for AAV Quantitation

    Type
    Blog Post
    Published
    Jan. 16, 2020, 2:18 p.m.
    ...benefit, replicates are built into the technology. One well (containing thousands of droplets) can be sufficient...fluorescence amplitude measured for each droplet in each well (Figure 2). A clean ddPCR should have a clear separation...negative (gray) droplets. The no template control wells should have very few positive droplets. In the image... copy per microliter in the no template control wells. You’ll also notice a reduction in the number of...cross contamination into a no template control (NTC) well is common. Here are some steps you can take to achieve...applications for the technology.  Droplet digital PCR is well suited for the detection of low copy numbers. For...used in the detection of rare sequences and single cell analysis. It is also being used in the detection...

  20. Plasmids 101: Colony PCR

    Type
    Blog Post
    Published
    May 12, 2016, 2:30 p.m.
    ... Orientation-Specific Tells you the orientation of the insert Tells you if the insert is present...product. Additionally, this type of primer only tells you if your specific insert is present but not if...without the insert. This type of primer pair can tell you if the insert is the correct size and whether...Insert-Specific Simple "yes or no" test results Doesn't tell you the orientation of your insert or if the insert...band (-) result as insert-specific primers but also tell you whether the insert has the correct directionality...clones you’ll need to send for sequencing, but won’t tell you if your products have any mutations.  There ...

  21. Analyzing CRISPR Editing Results with ICE from Synthego

    Type
    Blog Post
    Published
    May 8, 2018, 1 p.m.
    ...processing, the window shows a yellow checked circle. Typically, a yellow check mark indicates that a particular...score (KO-score) which represents the proportion of cells that have either a frameshift or 21+ bp indel. This...Following delivery of CRISPR components into target cells, genomic DNA from both edited and unedited (control...in front of that sample. You can hover over the yellow or red circles to gather details on the issues ... score. KO Score - Represents the proportion of cells that have either a frameshift or 21+ bp indel. This...vertical dotted line represents the cut site, and yellow + symbol on the far left marks the wild type sequence...

  22. CRISPR-mediated Plant Base Editors

    Type
    Blog Post
    Published
    Jan. 3, 2019, 1:35 p.m.
    ...homology-directed repair (HDR) pathway which occurs in cells at low frequencies and relies on the efficient delivery...Later the resultant U-G pair is either repaired by cellular mismatch repair machinery making an original C-G...editor. A massive protein engineering effort by Gaudelli et al. resulted in the successful evolution of...’ to ‘I’ (inosine), which is treated as ‘G’ by cellular polymerases. Subsequently, an original genomic... is converted to a G-C pair (Figure 1). As the cellular inosine excision repair is not as active as uracil...protospacer, ABE6.3 or ABE7.9 are better suited (Gaudelli et al., 2017). Our vectors have two Bsa1 sites...353.6305 (2016): aaf8729. PubMed PMID: 27492474. 3. Gaudelli, Nicole M., et al. "Programmable base editing ...

  23. How to Write a Scientific Review Article

    Type
    Blog Post
    Published
    Feb. 16, 2017, 3:30 p.m.
    ...a review article on histone modifers in B and T cells with my adviser when I was a graduate student. Naturally...aspect of the field you plan to review. If it’s a well-researched field, you may need to get specific to...these themes, and also for articles from the active/well-known researchers in the field. I made sure to find... find information from genome-wide studies, as well as results from smaller and more specific studies....studies. I also did not limit myself to the well-cited or popular papers, but looked for papers from a wide ... change the sentences, make it flow, and start telling the story. Like any editing process, you will need...acetyltransferases and histone deacetylases in B-and T-cell development, physiology and malignancy." Genes &...

  24. Antibiotic Resistance: An Old Solution but a New Problem

    Type
    Blog Post
    Published
    Nov. 10, 2016, 3:30 p.m.
    ... attack the cell wall of bacterial cells; however, Mycoplasma species do not possess cell walls. As a ...bacteriophage (viruses that only infect bacterial cells), or a bacterium picking up resistance genes from...determining drug resistance status, it is a very common, well-trusted method that is frequently used in the clinical...conserved, non-negotiable process within the bacterial cell, antibiotic resistance is very likely to develop...globally: final report and recommendations." London: Wellcome Trust & HM Government (2016). 2. CDC Information...

  25. Plasmid Preps: Different Purity, Different Quantities, Different Uses

    Type
    Blog Post
    Published
    June 16, 2020, 1:25 p.m.
    ...lipopolysaccharide of the cell wall of Gram negative bacteria like E. coli that affects tissue culture cell line viability...introduces transformation grade plasmids into a bacterial cell. Transformation grade DNA  Transformation is ...transformation of chemically and electrocompetent cells is minimal (picogram range). It can be extracted...Figure 3: Need to introduce plasmids into mammalian cells? Use transfection grade DNA. Transfection is ...process of inserting an external plasmid in mammalian cells and it will likely require micrograms of plasmid...

  26. Your Top Requested Plasmid in 2015!

    Type
    Blog Post
    Published
    Dec. 30, 2015, 3:30 p.m.
    ...both Cas9 and your gRNA of interest to mammalian cells. It was designed to have increased lentiviral titer...knock-out thousands of genes in mixed population of cells as part of pooled library screens (1,2). The human...pooled libraries targeting both mouse and human cells are avaliable for distribution. To learn more about... includes work probing mTOR signaling in cancer cells, deciphering the mechanism of matrix metalloproteinase...Genome-scale CRISPR-Cas9 knockout screening in human cells." Science 343.6166 (2014): 84-87. PubMed PMID: 24336571...Common Ways to Deliver CRISPR Plasmids to Mammalian Cells Resources on the Addgene Website Visit our Lentiviral...

  27. How Exercise Purges Stress and Keeps Depression at Bay

    Type
    Blog Post
    Published
    Nov. 25, 2014, 4:02 p.m.
    ...It’s well known that exercise can help fight stress and depression, and now researchers have reported... how it works. The findings in a recent issue of Cell demonstrate how PGC-1a1, a transcriptional coactivator... press release. “We actually found the opposite: well-trained muscle produces an enzyme that purges the... in a commentary about the new work published in Cell Metabolism. But, they note, there is plenty more...including psychiatric and cognitive conditions as well as neurodegenerative diseases.” I don’t know about...JR, Erhardt S, Lindskog M, Ruas JL. doi: 10.1016/j.cell.2014.07.051. PMID: 25259918 Muscle over mind. Moon...

  28. Keeping Up With CRISPR/Cas9

    Type
    Blog Post
    Published
    March 11, 2014, 5:55 p.m.
    ...Addgene Outreach Scientist and fellow blogger Caroline LaManna put it well: "Wow, custom mutations in monkeys...RNA-Mediated Gene Targeting in One-Cell Embryos. Niu Y, et al. Cell. 2014 January 30. (Pubmed) Programmable...correct disease mutations, both in mice and in human cells. But that important advance was quickly followed...  David S. Weiss of Emory University summed it up well in that New York Times feature when he said, “All...

  29. Five Popular Model Organisms, Part 2

    Type
    Blog Post
    Published
    Sept. 29, 2020, 1:15 p.m.
    ...By Justin Ng and Alyssa Cecchetelli Science is a constantly evolving and demanding field, requiring a...rats have some key differences in physiology, intelligence, and human correspondence that give them an ...routinely used in an array of neuroscience research (Ellenbroek and Youn, 2016). For these reasons rats were ...transgenic axolotls for an array of different studies. Elly Tanaka’s lab has constructed several vectors for...are often used to study vertebrate embryology and cell development. One major resource for frog research...Organism to Study and Model Human Genetic Disease. Cells Tissues Organs, 205(5–6), 303–313. https://doi.org...https://doi.org/10.7554/elife.50651 Vieira, W. A., Wells, K. M., & McCusker, C. D. (2019). Advancements to...

  30. SARS-CoV-2/COVID-19 Detection Methods Based on CRISPR/Cas

    Type
    Blog Post
    Published
    May 5, 2020, 1:15 p.m.
    ...activity which cleaves the supplied fluorescently labelled ssDNA substrates. Upon cleavage, the fluorescence...complementarity between the gRNA and a given fluorescently labelled substrate, gets activated and performs non-specific...strips to detect the cleavage of fluorescently labelled RNA, the authors employ a P51 cardboard fluorescence... with streptavidin coated beads. Fluorescently labelled dCas9 complexes containing sgRNA that have no ...Shravanti Suresh is a PhD candidate who investigates cellular aspects of CRISPR/Cas immune systems in the Sashital...2020.04.07.028167 Broughton JP, Deng X, Yu G, Fasching CL, Servellita V, Singh J, Miao X, Streithorst JA, Granados ...Biotechnology. https://doi.org/10.1038/s41587-020-0513-4 Kellner MJ, Koob JG, Gootenberg JS, Abudayyeh OO, Zhang...
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