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Showing: 401 - 420 of 1010 results

  1. Tips for Getting a Faculty Position

    Type
    Blog Post
    ...colleges, private universities, and medical schools. I will emphatically deny that I have a magic formula..." manual on applying for faculty positions in biomedical science. My motivation was to share my experiences...told all of the nuances of the research in as technical of language as possible. Seriously. My suggested... trying to say X." She would then look at me quizzically and ask why I didn't just say that? Bottom line...especially helpful. I set out to make something practical and accessible. If there's something you come ...the Janelia Research Campus of the Howard Hughes Medical Institute. His interests include the quality control...

  2. Plasmids 101: Expressing shRNAs for RNA Interference

    Type
    Blog Post
    ...structures that are processed by a ribonuclease called Dicer. In a great simplification of the process...pri-miRNA, which is then processed by a complex called Drosha to pre-miRNA. miRNA plasmids are engineered...backbone Choosing the promoter Mature siRNAs typically range from 19–22 nucleotides and are 100% complementary...’t just use the mature sequence in a plasmid and call it a day. You need to include both sides of the ...for quick in vitro experiments. shRNA plasmids typically use RNA polymerase III promoters, which transcribe... versatile gene-silencing machine. Journal of Biological Chemistry, 284(27), 17897–17901. https://doi.... DICER: structure, function, and regulation. Biophysical Reviews, 13(6), 1081–1090. https://doi.org/10.1007...

  3. Antibodies 101: Normalization and Loading Controls for Western Blots

    Type
    Blog Post
    ... why you might want to be careful relying on so called “housekeeping proteins.” Fully quantifying a western... later steps in this chain (blue) to infer or calculate things about earlier ones. In practice, multiple...popular choices for loading controls. These are often called “housekeeping proteins” and are detected with an... will almost certainly be overloaded. One paper called this problem “the most common error associated ...Data is presented as mean ± SEM. Adapted from Analytical Biochemistry, vol 440, Gilda, J. E., & Gomes,...loading control to β-actin for Western blots. Analytical Biochemistry, 440(2), 186–188. https://doi.org...Antibodies 101: The Basics of Western Blotting Technical Design of a Western Blot Troubleshooting and Optimizing...

  4. How to Negotiate a Successful Lab Start Up Budget

    Type
    Blog Post
    ...where you can start to identify which items aren’t critical to your research success. If you can get away ...resources to keep, and which ones to abandon. It’s critical to make these decisions in the following order... budget can help you compare the monetary and empirical value that helps you determine whether or not ...know how to decide which components are the most critical to your research, negotiations can be much easier...negotiating finances, instead of solely discussing numerical values, try to keep conversations focused on research...saving the institute time and energy that would typically be wasted going through some of the questions ...is your goal, then understanding your needs is critical to a successful negotiation and collaboration....

  5. Optogenetics + CRISPR, Using Light to Control Genome Editing

    Type
    Blog Post
    ...temporal and spatial control. Optogenetics uses genetically encoded tools, such as microbial opsins, to control...light-inducible heterodimerizing cryptochrome 2 (CRY2) and calcium and integrin-binding protein 1 (CIB1) proteins....this approach. This study generated the first optically active saCas9 protein. Stop light! Photoactive...different approach. These researchers used a genetically encoded photocaging technique to insert a light-removable...light-removable protecting group, specifically a nitrobenzyl photocaged lysine (PCK), on the Cas9 protein...Hemphill et al. presented data showing that this genetically encoded, photocaged Cas9 system could silence...editing and transcription. Label genomic loci Photochemical Control Hemphill et al., 2015   Nitrobenzyl-caged...

  6. CRISPR/Cas9 FAQs Answered!

    Type
    Blog Post
    ...it's usually more efficient than plasmid donor. Typical design is to have 50–80 bp of homology arms on ...changes (>100 bp insertions or deletions), we typically use plasmid donor, with two homology arms on each...transfection in cells like HEK, we use around 400 ng of a typical size (~5 kb) donor plasmid with Cas9/sgRNAs vector...plasmids. For ssDNA oligo design, the Zhang lab typically uses around 100–150 bp total homology. The mutation... giving 50–75 bp homology arms. The Zhang lab typically use PAGE purified long oligos. For large changes...100 bp insertions or deletions), the Zhang lab typically uses a plasmid donor, with two homology arms on...like HEK, the Zhang lab uses around 400 ng of a typical size (~5 kb) donor plasmid with Cas9/sgRNAs vector...

  7. Bringing Sustainable Practices to the Lab: Innovations

    Type
    Blog Post
    ...you’ve saved. Repurposing regulated medical waste Regulated medical waste (RMV), which we collect in those...scores products by their environmental impact on a scale of 1-10: the lower the score, the lower the environmental...companies to obtain these labels! Reusing plastic Historically, scientists washed and reused materials such...flow cytometers. These fluidic machines handle biological samples and are cleaned between each use. You...those red biohazard bags, typically is destined for a landfill. However, Triumvirate Environmental developed...

  8. What's New in CRISPR - December 2019

    Type
    Blog Post
    ...deaminase (TadA) to a catalytically impaired Streptococcus pyogenes Cas9. They called this editor Target-...-to-inosine (A-to-I) RNA editor called REPAIR by fusing catalytically inactivated RNA-targeting Cas13 ...plasmids available at Addgene. We will still periodically focus on specific CRISPR plasmid tools more ...editor still retains A-to-I editing. This system is called RNA Editing for Specific C-to-U Exchange, or RESCUE...

  9. CRISPR Protocol for Genomic Deletions in Mammalian Cell Lines [Video]

    Type
    Blog Post
    ...Regulatory DNA function is typically inferred based on correlative biochemical characteristics and ectopic...Canver of Boston Children’s Hospital and Harvard Medical School. Addgene is proud to present a video reprint...for deletion screening by conventional PCR. Biallelically disrupted cells can be easily identified. Even...investigator at Boston Children’s Hospital and Harvard Medical School. His lab studies the molecular genetics ...Matthew Canver is an MD/PhD student at Harvard Medical School in the laboratories of Stuart Orkin and ...

  10. Future of Research Conference - Remarkable Opening Session

    Type
    Blog Post
    ...Kristin Krukenberg and Jessica Polka from Harvard Medical School and a long list of supporters from all of...peer review and grant awards systems need to be radically overhauled.  Finally, the science publishing system...excellence to begin effecting change.  Panelists call for action Each of the speakers in the panel had...Director of the Office of Postdoctoral Affairs at UNC, called her presentation “Disequilibrium, Disillusion and...Associate Professor of Systems Biology at Harvard Medical School, titled her presentation “How to Train Your...and take action. More Reading: “Rescuing US biomedical research from its systemic flaws” by Bruce Alberts...

  11. CRISPR 101: Off-Target Effects

    Type
    Blog Post
    ...expression by various mechanisms. The RNA guide is typically 20 nts and the genomic locus must often be proximal...promiscuous. In other words, they can sometimes localize to unintended sites with sequence similarity to... the on-target site. When a CRISPR-Cas system localizes to unintended sites and performs its programmed...product with CRISPR to (hopefully) be used in human clinical trials. An off-target rate of 5% is far too high...Cas-OFFinder, CCTop, and many more. While such software typically only considers DNA sequence and doesn’t take into...deadCas activator/repressor experiments where only localization of the Cas occurs. 2 gRNA approach Off-targets...effectiveness as a control. Since off-targets typically confound an experiment due to their location (...

  12. Viral Vectors 101: Optogenetic Tools

    Type
    Blog Post
    ...Deisseroth, K. (2005). Millisecond-timescale, genetically targeted optical control of neural activity. Nature...Ganguli, S., & Deisseroth, K. (2019). Cortical layer–specific critical dynamics triggering perception. Science... activated tools, can control everything from electrical activity to gene expression, and even CRISPR ...through multiple intra-cellular signaling cascades. Optical methods for controlling GPCR activation have been...

  13. Plasmids 101: Restriction Cloning

    Type
    Blog Post
    ...that can be easily maintained in a variety of biological systems. Read on for an in-depth breakdown of...insert and 1μg of plasmid backbone. It is also critical that as much of the backbone plasmid as possible...step, depending on the phosphatase you choose. CIP (calf alkaline phosphatase) or SAP (shrimp alkaline phosphatase...base pairs for each varies, it is difficult to calculate this based on DNA concentration alone. One method...varying ratios of recipient plasmid to insert. It is critical to set up a negative control ligation reaction...electro-competent cells instead of the more common chemically-competent cells. You should perform, at minimum...purifications, you generally lyse the bacteria; add chemicals to precipitate out the high molecular weight genomic...

  14. SunTag and Fluorescent Imaging

    Type
    Blog Post
    ... then study where the protein is localized, and how its localization and expression may change across ...transcriptional activation. Multiple nuclear localization signals (NLS) increase protein import into the...technology, the Vale and Weissman labs took this biological lesson and created a system to amplify fluorescent...modified the GCN4 sequence to increase its alpha-helical structure and stability, creating v4 SunTag. Since... also affect protein activity, half-life, or localization, these concerns are greater with SunTag. v1 ...

  15. Addgene @ Keystone: Thoughts on Precision Genome Engineering and Synbio

    Type
    Blog Post
    ...poster tubes (or as our baffled flight attendant called them, "long, skinny things") and generally exuding... tales of back country skiing, fly fishing, and local grizzly bear attacks. He described one such recent...All Addgene depositors! Addgene was mentioned specifically in Dr. Zhang's introduction. His willingness...Cas9 that is improving the nuclease specificity dramatically and reducing off-target effects. Other young...discussed some of the many challenges of creating biological circuits, and I can only say this science is ... deposited and distributed – have changed so dramatically in the past 4 years. I kept busy for the whole...

  16. Antibodies 101: Epitope Tags

    Type
    Blog Post
    ...an antibody that works for your application, specifically detects your protein, is species compatible,...along with some pros and cons. FLAG FLAG is a synthetically derived 8 amino acid tag (DYKDDDDK) that can...possibly interfere with protein function and localization. If you need monoclonal GFP antibodies or guidance... N or C terminus of a protein may disrupt the biological function of some proteins. Protein folding issues... In rare cases, tags can also affect protein localization and solubility, so be on the lookout for aggregation...Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems. Appl Microbiol...

  17. What the HEK?

    Type
    Blog Post
    ...chromosome is present in this line, making it genetically female. HEKs are generally cultured as an adherent... cellular research tools have been optimized specifically for and in HEKs. These systems built around ...science research all the way up to the production of clinical viral therapies. Protein production  HEKs are...recombinant proteins at high levels. In a case where clinical applications of recombinant proteins are necessary...also grow in suspension, which allows for large scalability, and can also be adapted for serum-free growth..., which can make for a seamless transition of biological tools to other researchers using the cell line...

  18. The Strength of Story Telling

    Type
    Blog Post
    ...arguing with me. First I calmed the situation down by opening a dialogue specifically about the project and...We spoke about it for a while and he eventually calmed down, apologised and admitted he’d been under a...wherever it makes sense to start and try to be as chronological as possible — if you find yourself jumping backwards.... Your story should be too. Use drama Use any rhetorical technique you have in your arsenal to make sure...start point and it needs a good end point. One thematically pleasing option is to end at the present — ‘...

  19. Behind-the-scenes of the Isolation of the Thermostable IgnaviCas9 From a Yellowstone Hot Spring

    Type
    Blog Post
    ...Extremophiles - phylogenetically diverse microbes living in extreme physical and chemical conditions like...Stanford University became interested in exploring biological dark matter – large tracts of the microbial tree... to be diversity hotspots containing abundant biological dark matter and so the lab organized a sampling...like YNP thermal sites - are also of practical interest since they’ve previously yielded valuable enzymes...

  20. Photosensitizer Induced Cell Ablation with FAP-TAP MG-2I-dL5**

    Type
    Blog Post
    ...targeted cell ablation via ROS is achieved through localized expression of the FAP. A new photosensitizer:...the main advantages of MG-2I-dL5** over other genetically encoded photosensitizers is that it works with... ROS because NIR light doesn’t excite natural biological chromophores found in cells.  He et al also show...compared MG-2I-dL5** head-to-head with KillerRed, a genetically encoded red fluorescent photosensitizer protein...Watkins, S. C., . . . Bruchez, M. P. (2016). A genetically targetable near-infrared photosensitizer. Nature...Chepurnykh, T. V., . . . Lukyanov, K. A. (2005). A genetically encoded photosensitizer. Nature Biotechnology...
Showing: 401 - 420 of 1010 results