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We narrowed to 812 results for: APP

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  1. Custom CRISPR Screens & the Green Listed Software

    Type
    Blog Post
    Published
    July 11, 2017, 2:30 p.m.
    ...genes provided by the user. This approach is useful for several applications as will be discussed below. The... screen approach is to use smaller libraries targeting subsets of genes. With such an approach, you lose...custom CRISPR screen and the appropriate phenotypic readout, similar mapping could be rapidly performed ...challenges with a custom screen approach is to identify an appropriate list of genes to target. Thus, ... involved in a studied cellular behavior. This approach is particularly suitable for in vivo screens, ...drugs. Known drug targets  Another interesting approach is to design screens against known druggable targets...preferentially those for which validated inhibitors or even approved drugs already exist. If you are lucky and find...

  2. Magnetic Control of Proteins: More than a Dream

    Type
    Blog Post
    Published
    Sept. 24, 2024, 1:15 p.m.
    ... tools for familiar applications. But how does one invent a tool for applications that don’t even exist...and develop potential applications.  Andrew is a frequent collaborator and is happy to give advice and support...phenomenon, improve the tool, and develop new applications for magnetoresponsive proteins. Motivated by...organisms) are opaque, which makes most biomedical applications of these tools difficult or impossible. But ...tool with magnets would enable new research and applications that don’t yet exist, but it wasn’t clear that...molecules like proteins with magnetic fields generally appears to be weak — if they exist at all. Andrew and Maria...fluorescence intensity decreased whenever the magnet approached the sample (by less than 1%) and recovered after...

  3. Designing Your Chalk Talk for the Academic Job Interview

    Type
    Blog Post
    Published
    March 12, 2019, 1:37 p.m.
    ...single model. The first approach can lead to new directions, while the second approach could terminate with... Many thanks to our guest blogger, Erik Snapp! Erik Lee Snapp received his PhD in Molecular Microbiology...This post was contributed by Erik Snapp, the Director of Student and Postdoctoral Programs at the Janelia.... Given this large sum, one might expect that applicants would work for months to hone a chalk talk. Yet...preparing for both types. Your research vision is your approach to resolving a significant knowledge gap in your...has unclear utility. You cannot predict what will happen. Without the prediction, you have no hook. A battery... you need to be able to describe experimental approaches in terms of the question, the methods, the tools...

  4. Avoiding the Dark Side of Fluorescent Protein Fusions with mOX FPs

    Type
    Blog Post
    Published
    Oct. 27, 2015, 3 p.m.
    ...our guest bloggers Erik L. Snapp and Lindsey M. Costantini! Erik Lee Snapp received his PhD in Molecular... post was contributed by guest bloggers Erik L. Snapp and Lindsey M. Costantini. "You underestimate the... of a mystical "Force," this quote is equally applicable to many microscopy experiments with fluorescent...not fluoresce. A dark population is not readily apparent and can confound quantitative imaging experiments...for use in cytoplasmic environments. However, approximately 40% of human (and most eukaryotic) proteins ...have reported that FP cysteine residues form inappropriate disulfide bonds in the secretory pathway (Figure...localization. For example, the formation of inappropriate inter-chain disulfide bonds between FPs blocks...

  5. A Guide to Getting Started in Undergrad Research

    Type
    Blog Post
    Published
    June 8, 2021, 1:15 p.m.
    ...developed and applied. The current pandemic serves as a striking example of the applicability and importance...research. I applied, hoping that the experience would improve my scientific and application writing skills...science (as opposed to translational, biomedical, or applied sciences), people conduct research to answer questions...revelation. Science in any field can generally be applied to improve our lives and solve global problems....grants Varying degrees of engagement with what happens in the lab Official title is typically Professor...the people around you. Be proactive Rather than applying only to posted job offerings, the best research...ties into the previous one too. I would recommend applying for multiple positions—don’t put all your eggs...

  6. Advice on Career Paths and the Green Card Process for International Researchers and Entrepreneurs

    Type
    Blog Post
    Published
    Dec. 20, 2017, 2:33 p.m.
    ...received 116,224 employment based green card applications and approved 91,023 during the first 3 quarters of ...options discussed in this post benefit from the applicant thinking about the process early, keeping good...is a numerical quota of 85,000 total H-1B visas approved each fiscal year: 65,000 visas plus 20,000 reserved...occupation" as requiring theoretical and practical application of a body of highly specialized knowledge in ... April 1 deadline for companies to submit an application on their behalf.  If an individual obtains a ... cap subject H-1B (last year less than 30% of applicants received sponsorship) then he or she can start...1B petitioners are exempt from the cap if the applicant works at institutions of higher education, a related...

  7. Evolution of Lab Techniques

    Type
    Blog Post
    Published
    June 21, 2016, 2:30 p.m.
    ...real-time PCR (4). Restriction mapping and modern sequencing Restriction mapping informs on DNA sequence through...useful applications and have informed many current techniques. For example, restriction mapping led to...historical techniques: southern blotting, restriction mapping, and sequencing gels, as well as their modern equivalents...comparison to a control sample. Southern blotting applications range from confirming the results of cloning...gene that causes sickle cell anemia. The mutation happens to destroy a sequence recognized by the restriction... was commonplace, researchers used restriction mapping to investigate DNA sequence content by comparing...of the DNA. Starting in the 1970s, restriction mapping was commonly used to map DNA and examine genomes...

  8. Advancing Biology with Zebrafish: Genetic Tools for Developmental Studies and More

    Type
    Blog Post
    Published
    Aug. 13, 2019, 12:58 p.m.
    ...zebrafish embryo has been mapped using single cell RNA sequencing approaches (Harland, 2018). Several ...investigations. Genetic resources for zebrafish A common approach to unravel the gene networks that drive biology...DNA into the genome is useful for a variety of applications. For example, specific tissues such as blood... regions is also possible using an alternative approach that exploits the bacteriophage integrase phiC31...useful, I’ll outline two recent lineage tracing approaches that led to important insights in developmental...tracked using microscopy and advanced computational approaches (Keller et al., 2008). This microscopy-based ...pathways control this process. Additional zebrafish applications and conclusions Zebrafish are also popular outside...

  9. Important Considerations in Optogenetics Behavioral Experiments

    Type
    Blog Post
    Published
    Oct. 1, 2015, 2:30 p.m.
    ...subsequent, repeated activation), this is an approximation of reward or the sense of pleasure that the ...EthoVision. Consult the literature to find all the applications of optogenetics relevant to your field. Ultimately...behavior, certain technical considerations still apply. Testing stimulation First, In lieu of electrophysiology...stimulate them for 30min to an hour and observe what happens. You never know how stimulation of a particular...optimization, turn on the laser and watch what happens. Who knows, you may observe something that could... discovery. Monitoring behavioral output The appropriate behavioral readout for your optogenetics experiment...brain region that may be important in memory, an appropriate behavioral test might be contextual fear conditioning...

  10. 15 Hot Plasmids from 2017

    Type
    Blog Post
    Published
    Jan. 9, 2018, 3:02 p.m.
    ... a bioinformatics approach examining genomes possessing CRISPR systems with apparent self-targeting. Two... plasmid or group of plasmids' functions and applications. We hope that these articles make it easier ...includes four promoters, six fluorophores with nonoverlapping emission spectra (N- and C-terminal tags for...Targeting and manipulating interneurons using a viral approach Genetically modified organisms like transgenic...successfully targeted via viral particles, an effective approach to target specific neuronal subtypes was not available..., Gordon Fishell’s lab has developed a new AAV approach using regulatory elements that restrict expression...tools in virtually any vertebrate. This novel AAV approach allows you to target and functionally manipulate...

  11. How to Deposit Your Plasmids with Addgene

    Type
    Blog Post
    Published
    Feb. 27, 2024, 2:15 p.m.
    ... matches to your insert. Select the appropriate gene if it appears in the Entrez Gene list, as this will...would search for it in PubMed and choose the appropriate publication. This publication will be listed ...preprint. In the case of a preprint submission, we are happy to update the publication information once the manuscript...pass our standard QC process and the MTA has been approved. The spreadsheet option is recommended for ...contain epitope tags or fusion proteins that are appended to new inserts through cloning. *Note — If you...complete these pages sequentially by entering the appropriate data and pressing "Save and Continue to Next ...around to the different pages by clicking on the appropriate boxes at the top of the page. Pro Tip! Be sure...

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

    Type
    Blog Post
    Published
    April 16, 2024, 1:15 p.m.
    ...the filament from (a-b), showing the appearance and disappearance of 649-mDia1 or 549-CP (upper, middle...block its catalytic activity and instead become trapped in a covalent bond with its substrate. Then, all...having to clone a whole new construct for every application. Plus, the photophysical properties of chemical...advantages over fluorescent proteins, opening up new applications.   Figure 1: Live-cell labeling strategies...acids 1 Various Many Complicated   Applications Organic dyes are typically both brighter and...self-labeling tag and a fluorescent protein) enables applications like multi-color imaging (Figure 2). If you’...Actin, cyan), with formin (649-mDia1, magenta) and capping protein (549-CP, yellow) at the tip. 649-mDia1 ...

  13. Transferable Skills Guide: Cross-team Communication

    Type
    Blog Post
    Published
    Nov. 21, 2019, 1:53 p.m.
    ...depending on the conference you’re attending and who approaches you. You might also have cross-team projects ...communication. Although these real world examples did not happen in the lab, they have parallels to collaborative...types or purposes of each given container, when applicable Any other relevant information Prescribing an...misinterpretation, that misinterpretation was definitely happening. Our solution for this problem was the creation...lab setting, where student and postdoc turnover happens regularly and it behooves everyone in the lab to...potentially ambiguous terms. Defining words and applying annotation can be particularly useful in academic.... But if you implement it well, here’s what can happen: More effective meetings. When everyone at a meeting...

  14. Selecting Your Plasmid Purification Kit

    Type
    Blog Post
    Published
    May 27, 2025, 1:15 p.m.
    ... use one of two common approaches — columns or magnetic beads. Both approaches involve binding the plasmid... plasmid preps are the same. Your downstream application will dictate how much DNA you need to recover...recover and the level of quality required. Some applications require very little DNA and you can get away with...with slightly lower quality preps, while other applications require large amounts of high quality DNA. Figure... a High Quantity/Low Quality mindset for any applications. DNA quantity is fairly self explanatory and...possibility of damaging the plasmid DNA itself, which can happen if you leave the sample in a lysis buffer for too... the DNA from the substrate. In a column-based approach, the substrate is a silica matrix fixed in a column...

  15. An Inside Look into Addgene's Dev Team

    Type
    Blog Post
    Published
    Jan. 23, 2020, 2:45 p.m.
    ...truth is, there is a lot of software development happening at Addgene under the hood. A significant portion...this can lead to wildly different ideas of how to approach a problem, but it is these very differences that...similar cognitive biases and this would become apparent in the software we develop. We would all have ...framework is based on an iterative and collaborative approach to development. Scrum teams plan and break up ...are also able to consider different technical approaches that may come up during discussion.  One example...perspective which in turn helps us decide how to best approach it in our code.  Sometimes while thinking through...that the clarifications lead to the story being scrapped or broken up into smaller stories. Since this ...

  16. Getting the Most from Your Lentiviral Transduction

    Type
    Blog Post
    Published
    April 7, 2016, 2:30 p.m.
    ...virus If having happy cells is the first step of a successful lentiviral infection, happy virus is the second... express a gene of interest. While the general approach for using lentivirus, infect and select, seems... cell lines, ELISA, fluorometry, or PCR-based approaches to detect even low levels of contamination. For...Uphoff et. al. outline a do it yourself PCR-based approach complete with freely available positive controls...old. Ensuring that you are working with healthy, happy cells is one of the easiest ways to improve both...envelope. One drawback of the multiple harvest approach is that it tends to result in a higher volume ... freshly collected lentivirus for downstream applications. While this ensures that the highest titer virus...

  17. Expanded Access to Rabies Plasmids

    Type
    Blog Post
    Published
    Feb. 1, 2024, 2:15 p.m.
    ...non-refundable fee is applied to cover processing costs. Once the application is approved, Addgene then ships...excited to announce we now offer an export license application for rabies materials! Some of the materials in...international requestors. We also offer export license applications for VSV and SARS-COV-1 plasmids. Export control...license process    An export control license, once approved, allows Addgene to ship plasmids or viral vectors...will need to provide a brief description of the applicable research project when purchasing export-controlled...export-controlled materials. Addgene will then apply for the license for distribution to the requester’s organization...offered VSV and SARS-COV-1 plasmids under this application process since May 2020. We find this process ...

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

    Type
    Blog Post
    Published
    June 3, 2021, 1:15 p.m.
    ...led to their application for basic and translational research.  Basic research applications of scFvs: ...blood clearance, which is useful for imaging applications; better tissue penetration which is useful for... for therapeutic and imaging applications; and reduced immunogenicity when administered in vivo due to...from blood can be a drawback for therapeutic applications where longer retention times often increase ...laborious.  To avoid this, one or more of these approaches are often used for scFv expression in bacteria...since it’s genetically encoded.   Translational applications of scFvs: Just like antibodies, there are many...2019).Many of the scFvs designed for oncology applications are reformatted as bispecific molecules which...

  19. Antibodies 101: Selecting the Right Antibody

    Type
    Blog Post
    Published
    March 15, 2022, 1:15 p.m.
    ...fits all When choosing an appropriate antibody first look at the application-specific data provided on...your specific application. Consider also that there may be variations within an application. Take immunohistochemistry... a linear antigen tend to work very well for applications that denature a protein to its primary (linear...while the website highlights a variation of your application, a publication successfully used it in a similar...plan to. To secondary or not to secondary?  The application will also determine whether you should use an... as it will help to amplify the signal. Some applications use either direct (primary-conjugated) or indirect... are secondary considerations to validation, application specificity, and experimental considerations ...

  20. CRISPR 101: Off-Target Effects

    Type
    Blog Post
    Published
    Feb. 8, 2024, 2:15 p.m.
    ...many applications, it is the only comprehensive method available. Targeted sequencing approaches More...differently. For a comprehensive approach, RNA-seq would be an appropriate method for an activator or repressor...lower them even further. While not all of these approaches will work for every experiment, at least one ...where only localization of the Cas occurs. 2 gRNA approach Off-targets that come from mutations arise from...More targeted alternative sequencing approaches have been developed. Cas ChIP-Seq, where only DNA sites ...especially when DNA mismatches are involved. Two other approaches have been used - GUIDE-Seq and integrase-defective...site-specific detection downstream. The con to these approaches is that NHEJ must perform the repair to allow...

  21. Antibodies 101: Choosing the Right Isotype

    Type
    Blog Post
    Published
    April 6, 2023, 3 p.m.
    ...your experiment (all well validated for your application, of course!) When the stars are aligned and the...factors that affect isotype choice. Target and application should be considered The first question you may... a strong signal and to work in a variety of applications. The desired specificity of your secondary antibody...affect my experiment?” Well, it depends on the application and target. If you are running a basic western...target is expressed at very low levels or your application requires a high degree of affinity, then species...considered when choosing an antibody. In some applications probing species-on-species, i.e. using a mouse...easily distinguishable from each other. In a direct approach, that is simple - but what about when you need...

  22. Addgene is Expanding Our Viral Vector Service!

    Type
    Blog Post
    Published
    Oct. 15, 2024, 1:15 p.m.
    ... to be placed immediately upon request approval. Once approved, the request will remain in your account... in this blog post.     When will the expansion happen?  You will be able to request Packaged on Request...request will be reviewed by Addgene for legal approvals and technical compatibility with our production...process will take 1–2 days, and we anticipate approving the vast majority of requests.  If the vector ... request process.  Ordering If your request is approved, you will receive an email notification that your...is ready to order. An “Add to Cart” button will appear with the request in your Addgene account.  Orders...viral preps will be available at launch.  What happens after I order?  Once an order is placed, the Addgene...

  23. 22 Hot Plasmid Technologies from 2014

    Type
    Blog Post
    Published
    Jan. 6, 2015, 5:21 p.m.
    ...distinction of neighboring cells in imaging applications and the mapping of neuronal projections to their associated...while minimizing confounding secondary effects? An approach described as “knocksideways” (a British idiom ...domains, such as FKBP and FRB. The cell does not appear to mind extraneous proteins bound to its outer ...protein from expression to degradation. Chemical approaches may require expensive peptides or small molecules...and Technology Austria, decided to take a novel approach to control for these effects, drawing on the rapidly...optogenetics. Using a rational protein engineering approach, they designed Opto-RTKs, which activate signaling... on exposure to low-intensity blue light. This approach relies on the incorporation of light-oxygen-voltage...

  24. Finding Your Science Policy Path

    Type
    Blog Post
    Published
    Sept. 22, 2020, 1:15 p.m.
    ... can happen at the national level within the executive branch or in Congress, or can also happen at the... there will be posts about virtual meet ups and happy hours that you can attend to help build relationships...arriving here in DC, is that so much policy actually happens outside of the government itself. You can work ...The “little p” policy is non-governmental work happening at large organizations who have to develop policies...who decides how they are going pursue regulatory approval for a new product or a well-endowed philanthropy...Program to learn more about where science policy happens.  When do you do science policy? Many science ...Project for some example proposals and consider applying for their accelerator for some hands-on guidance...

  25. Negotiating Work and Life: How to Find the Joy

    Type
    Blog Post
    Published
    March 20, 2014, 3:04 p.m.
    ...sense and appreciate this – and they will be happy too. On the flip side, if you are not happy at work,... If you are happy at work, you will find balance. If you enjoy working and come home happy in the evening...actions we can take to help ourselves can be an appropriate first step. Check out Joanne's Reddit AMA Managing...turns going; that way you can have someone there clapping more often than not. You want your family and ...tools and tactics to plan your work and make this happen. Be realistic and: Your co-workers will learn ... about their safety when they leave home which happens very, very fast! I will never forget the time my...figure out a way to keep being ourselves and to be happy. Building your plan to live a good life may be the...

  26. Overcoming the AAV Size Limitation for CRISPR Delivery

    Type
    Blog Post
    Published
    Sept. 16, 2020, 1 p.m.
    ... are approximately 4.1 kb in length, allowing them to be packaged in AAV.   Split AAV approaches  While...germline genome editing, but in vivo gene editing approaches have been limited by problems with Cas9 delivery...packaging capacity of AAV (~4.7 kb). While this approach has been proven feasible, it leaves little room...potent Cas9 protein, the Zhang lab took a different approach. They analyzed over 600 Cas9 orthologs and found...be divided into two groups: longer orthologs approximately 1350 amino acids in size, which includes SpCas9...SpCas9, and shortern orthologs approximately 1000 amino acids in size. From the pool of shorter orthologs...effects of SpCas9 and SaCas9, Zhang’s group used an approach called BLESS (direct in situ breaks labeling, ...

  27. Hot Plasmids - November 2023

    Type
    Blog Post
    Published
    Nov. 14, 2023, 3:35 a.m.
    ...platform offers a promising approach for experimental therapies and medical applications.  Find DIRECTED plasmids...Figure 1: Choosing the best PE6 variant for your application. Image reused form Doman and Pandey et al., 2023...REcipient Cells Through Envelope Design). This modular approach combines an interchangeable set of fusogenic and... CreVLPs, and eVLPs for in vivo and in vitro applications.   Figure 2: Modular features of DIRECTED...interactions or map a local proteome, but some applications have been challenging or inaccessible. The Ting...  Qin, W., Cheah, J.S., et al. (2023). Dynamic mapping of proteome trafficking within and between living... from different promoters, specific time points appear as fluorescent bands in the fiber, resembling a...

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

    Type
    Blog Post
    Published
    May 5, 2020, 1:15 p.m.
    ...fluorescence can be visualised. Another approach was to apply the same concept to lateral flow strips ... Suresh from Iowa State University. Since its appearance, SARS-CoV-2 has spread to almost every part of... required: 37°C heat block and 62°C heat block Approach: Viral RNA is extracted and subject to isothermal... required: 37°C water bath and 42°C water bath Approach: RNA is extracted from a sample and subject to...cleaved. Cleavage results in fluorescence and this approach can be incorporated into a lateral-flow readout...blue light illuminator or UV light illuminator. Approach: Highly similar to the SHERLOCK diagnostic test...microliter Time: Not mentioned in the paper, likely approximately 1-2 hours Equipment needed: Mini PCR machines...

  29. Antibodies 101: Introduction to Immunofluorescence

    Type
    Blog Post
    Published
    Nov. 16, 2021, 2:15 p.m.
    ...is an application in which antibodies that have been joined to fluorescent molecules are applied to a ...immunofluorescence There are a couple different approaches to be familiar with when talking about IF, direct...BioRender.com   Indirect IF is a much more common approach than direct IF, so we will focus on this technique... Primary Antibody This is the first antibody applied to your sample, which will bind to your target ...Unsurprisingly, this is the second antibody you apply, which binds to the first antibody and is conjugated...distribute antibodies validate them in a variety of applications, including IF (or IHC or ICC), and will share... time to research antibodies, protocols, and appropriate controls and you will be well on your way to ...

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

    Type
    Blog Post
    Published
    April 8, 2025, 1:15 p.m.
    ...sample of your protein of interest (POI). This approach is reliable, but it’s overkill in many cases. ...than “condition 2.” By eye, the loading control appears about the same between the two conditions. But ...detected with an antibodies, just like the POI. This approach is so common that we’ve used it for our cartoon...cells. This means that at sample concentrations appropriate to detect a less-abundant POI, the loading control... control aren’t too far apart, they may have overlapping linear detection ranges, allowing you to accurately..., 2008; Gilda & Gomes, 2013). Depending on the approach you choose, your loading control measurement will...on the same gel or membrane (depending on your approach) as your final blot. Quantifying the entire lane...

  31. Plasmids 101: Methylation and Restriction Enzymes

    Type
    Blog Post
    Published
    June 30, 2016, 2:30 p.m.
    ...Blocked by overlapping methylation Not affected BsaI Not affected Blocked by overlapping methylation...Blocked by overlapping methylation HpaI Not affected Not affected Blocked by overlapping methylation...Blocked by overlapping methylation Not affected Not affected MscI Not affected Blocked by overlapping methylation...Not affected Blocked by overlapping methylation XbaI Blocked by overlapping methylation Not affected...methylation Not affected ClaI Blocked by overlapping methylation Not affected Not affected DraI Not affected...

  32. Is this the right place for me? 8 tactics for choosing a lab

    Type
    Blog Post
    Published
    Oct. 2, 2018, 12:56 p.m.
    ...think “but that won’t happen to me, I have a backbone”. The sad truth is, if it happened before, it almost...choosing a lab, but almost all of these guidelines can apply to any workplace or job. It amazes me when the most...scheduled or only ad hoc? Does the mentor keep appointments with group members or are they frequently cancelled...conflicts in the lab? Do they motivate with the appropriate balance of encouragement and criticism? Do they...was told that the PI removed a woman from the appropriate first authorship position on a publication of...and profoundly regretted it when the same thing happened to her a year later. I hear too many science trainees...almost certainly will also happen to you. As you speak to lab members and alumni, don’t ever disregard warning...

  33. Transferable Skills Guide: Public Speaking

    Type
    Blog Post
    Published
    April 20, 2023, 4 p.m.
    ...t just happen to you or me (though, let's be clear, they have all happened to me.) They happen to everyone...is, when it happens to other people, it’s normal and quickly forgotten, and when it happens to you, it’...communicator, I’ve done a lot of public speaking, and I'm happy to share what I've learned along the way. Own your...comes to public speaking, Murphy’s law definitely applies: what can go wrong, will go wrong. The trash removal...interest incorrectly on slides 8-16. Your notes disappear. The wrong version of your slide deck gets uploaded...completely normal to be anxious, nervous, and/or apprehensive. I still get nervous quite regularly, and I'...

  34. 27 Hot Plasmids from 2016

    Type
    Blog Post
    Published
    Dec. 22, 2016, 3:03 p.m.
    ...AID an appealing tool for studying essential proteins. Until recently, it was challenging to apply the AID-tagging...brief articles highlight the main features and applications of a partiular plasmid or set of plasmids. We...December 2016 (Q3) MARCH 2016Enhancer activity mapping with STARR-seq   Although many technologies have...classical TANGO assay, Kroeze et al. developed a new approach termed Parallel Receptor-ome Expression and Screening... (PRESTO-TANGO) to screen the NCC-1 library of approved drugs against the entire kit; a parallel analysis...multiple expression systems before they achieve the appropriate expression level for their gene of interest. ...GMAP-compatible vectors for a wide variety of biological applications. First, GMAP-compatible backbones for lentivirus...

  35. CRISPR in the Clinic

    Type
    Blog Post
    Published
    Nov. 2, 2023, 1:15 p.m.
    ... drug approval boards are being finalized, and it is very likely the treatments will be approved for use...disease. Meanwhile, Hans Clevers and colleagues applied CRISPR to disease correction in adult stem cells...CRISPR-based editing therapies that are currently approved or have made significant progress in clinical ...Therapies Cancer and CAR-T therapy The FDA has already approved CAR-T immunotherapy for cancer treatment by traditional...These therapies are delivered with an autologous approach, editing patient cells ex vivo and then returning...but especially concerning when using a systemic approach. Regarding effectiveness, if the editing frequency...meaningful way. Delivery, especially for systemic approaches, has its own unique set of obstacles. Thus far...

  36. FLEx Technology and Optogenetics: Flipping the switch on gene expression with high spatial and temporal resolution

    Type
    Blog Post
    Published
    Oct. 17, 2019, 12:28 p.m.
    ...which subpopulation of DA neurons could activate appetitive rather than aversive stimuli. In a recent paper...rAAV FLEx-based vectors to answer this question, mapping and characterizing the activity of the DA neurons...pursue a teaching career, and she now is a very happy scientific communication manager at genOway.     ... cell types for imaging and long-range circuit mapping." Journal of Neuroscience 28.28 (2008): 7025-7030...Philippe Coulon, and Ilka Diester. "Optogenetic approaches to study the mammalian brain." Current opinion...Yizhar, and Karl Deisseroth. "The development and application of optogenetics." Annual review of neuroscience...Mei, Yuan, and Feng Zhang. "Molecular tools and approaches for optogenetics." Biological psychiatry 71.12...

  37. Troubleshooting and Optimizing a Western Blot

    Type
    Blog Post
    Published
    Sept. 17, 2024, 1:15 p.m.
    ...Alternatively, you can run a reagent gradient. In this approach, you’ll run the same protein concentration in ...concentration of your reagent. Be aware that this approach requires excellent attention to detail, since ...down to voltage and time. Make sure to load an appropriate protein ladder on your gel and keep an eye on...too high. If you’re seeing a smiley face at an appropriate voltage (usually 10–15 V/cm of gel), your gel...box. If overheating remains an issue, which can happen with large proteins, try lowering the voltage and... buffer — so it’s better to start over if that happens.  Finally, if you’re seeing a weird issue and can...your reversible staining method of choice is appropriately sensitive. If using a PVDF membrane, you can...

  38. When is a Monomer not a Monomer? The Top Three Ways Your Favorite Fluorescent Protein Oligomerizes in Cells

    Type
    Blog Post
    Published
    April 19, 2016, 2:30 p.m.
    ...Many thanks to our guest blogger Erik L. Snapp! Erik Lee Snapp received his PhD in Molecular Microbiology...This post was contributed by guest blogger Erik L. Snapp. Stop using EGFP/GFP for fusion proteins! Despite...problem for fusion proteins. One of the major applications of FPs is to visualize the localization, dynamics... dimerize can form dynamic contacts that bring apposing membranes together and can wrap the membranes ...the Endoplasmic Reticulum and live cell imaging approaches. Erik is also an avid long distance runner, gardner... gardner, and cook.   References 1. Snapp, E.L., et al., Formation of stacked ER cisternae by low affinity...An experimental study of GFP-based FRET, with application to intrinsically unstructured proteins. Protein...

  39. CRISPR 101: Mammalian Expression Systems and Delivery Methods

    Type
    Blog Post
    Published
    Sept. 24, 2015, 2:30 p.m.
    ... the appropriate CRISPR tools for your application: Wildtype Cas9 or Cas9 nickase are appropriate for ...well as suitable applications. Expression System Components of System Application Mammalian expression...CRISPR mammalian expression systems, the typical applications for each, and potential delivery methods.   ...mutations near the gRNA target site. Choose an appropriate expression system and delivery method: Do you...electrophoresis and/or next-generation sequencing more appropriate? If you already have an end product in mind...or more sgRNAs and Cas9 are required for any application. If you want to include a homology directed repair...

  40. CRISPR 101: Ribonucleoprotein (RNP) Delivery

    Type
    Blog Post
    Published
    Sept. 6, 2018, 12:02 p.m.
    ...amenable to transfection or viral transduction     Appropriate promoters must be chosen for both Cas9 and gRNA... Cas9-gRNA ribonucleoproteins? One alternative approach, which avoids many of these complications, is ...used for most of the current genome engineering applications of CRISPR including: generating single or multi-gene...pathways. This makes Cas9 RNPs useful for CRISPR applications where limited expression of Cas9 is required...vendor, this can often be expensive. An alternative approach is to express and purify His-tagged Cas9 from ...this technique in cell culture, it has also been applied to genome editing of mouse zygotes, through a process...inner ear (Zuris et al. 2015). A more targeted approach using RNPs has recently been described in vitro...

  41. MXS Chaining

    Type
    Blog Post
    Published
    Feb. 7, 2017, 3:30 p.m.
    ...engineer various constructs with applications in flow cytometry approaches in mammalian cell culture systems...develop diverse fluorescent reporters is a useful application of high-throughput cloning. MXS Chaining is one...plasmids for fluorescence imaging or flow cytometry applications in mammalian cells. The modules used in this...transgene expression, or a variety of other clever applications. In one example, modular constructs generated...methods are suited for particular downstream applications. The tradeoffs between various cloning methods...from them. These enzymes might not, however, be appropriate for cloning genes from organisms with high CpG...quickly, and simply. We hope that you can similarly apply MXS chaining to your experimental needs. Let us ...

  42. CRISPR Between the Genes: How to Experiment with Enhancers and Epigenomics

    Type
    Blog Post
    Published
    Aug. 9, 2016, 2:30 p.m.
    ...structural proteins that make it possible, see the appendix DNA is a 3D fractal). Enhancer biology is a complex...acetylation) - tri_methylation In the next section we’ll apply the above formula to a real-world experiment. We...: the enhancers! This leads us to a three-step approach for designing an experiment to identify the critical...therapeutics. We’ll have more to say about clinical applications of CRISPR in the next section. In order to denote...mechanism for CRISPR. Therefore if a gRNA drops or disappears over time, we infer that the enhancer it targets...own CRISPR screens for enhancers, check out the Appendix. Conclusion: Shedding light on dark matter Precise...is the foundation of precision medicine. We can apply this knowledge to create targeted disease therapies...

  43. A Career in Grants: How To Become a Grant Professional

    Type
    Blog Post
    Published
    Aug. 20, 2015, 2:30 p.m.
    ...the candidate application process, the candidate’s ability to write a grant application through a written...funding opportunity coincided, and they stepped up to apply for a grant which lead to their new career path.... much more than writing. In fact, many grant applications and grant makers have moved away from long written...Now they require grant seekers to use online applications with significant character limits for the written...written portion of the grant application. They have increased their demand for stronger budgets with clear...duties can range from coordinating the grant application and submitting it, to evaluating the capacity...grant readiness, funding opportunity research, application writing and development, project implementation...

  44. Bacterial Toxin-antitoxin Systems as Molecular Biology Tools

    Type
    Blog Post
    Published
    Nov. 1, 2018, 12:35 p.m.
    ...activity of CcdB. Applications of ccdB in plasmid cloning Perhaps the most widely used application of CcdB is...used as biological tools for a wide range of applications in molecular biology or in vivo studies. Let...types of plasmid addiction systems and their applications. The discovery of toxin-antitoxin systems Toxin-antitoxin... by binding the GyrA subunit of DNA gyrase and trapping it in the cleaved complex. This results in DNA...construct. Other toxin-antitoxin systems and their applications MazE/MazF Aside from CcdB/CcdA, there are many...or diluted as the cell grows resulting in the appearance of unmethylated sites in the chromosome. The ...were retained in 75% of cells. They were able to apply this system as an orally administered diagnostic...

  45. Working 9-5: an Addgene Co-op

    Type
    Blog Post
    Published
    June 2, 2022, 2:15 p.m.
    ... QC/QA team as a DNA Sequencing Intern. The application process When I first saw the comprehensive job...for this co-op, I was immediately interested in applying. This had stood out to me because it gave me a...immediately clear that Addgene wanted to ensure applicants understood what the expectations were for the...the intended position. After submitting my application, I was contacted to participate in a brief fifteen...Friday, I received the official offer. I was so happy to hear back, I accepted that day and was excited...the depositing labs intended. With the use of applications like SnapGene and Geneious, I was able to determine...they would be disregarded - everyone was very approachable.     Fig. 1: Examples of DNA sequencing...

  46. Site Directed Mutagenesis by PCR

    Type
    Blog Post
    Published
    Aug. 2, 2016, 2:30 p.m.
    ...substitutions (or deletions) in a tailored manner. The approach can be used in conventional cloning (to introduce...introduce or remove restriction sites), in mapping of regulatory elements (to mutate promoters/enhancers in...of CRISPR; site-directed mutagenesis generally applies to plasmids, but may also facilitate genome editing... then, when the plasmid is digested with the appropriate restriction enzyme and run on a gel, one of the...ablates a restriction site, digestion with the appropriate enzyme will merge two smaller bands visible in...larger band (not shown). As a footnote; a similar approach can be used to identify Cas9/CRISPR-induced genome...template for the mutagenesis reaction. Alternative approaches to site directed mutagenesis Point-mutagenesis...

  47. Why Add Sucrose? Improved Yields for Adeno-associated Virus Preparation

    Type
    Blog Post
    Published
    Dec. 13, 2018, 1:14 p.m.
    ... AAV2 yields approximately 1.8-fold without affecting cell viability. This effect appears to be serotype...AAV yields. Given the wide range of downstream applications scientists rely on our AAV preparations for,...assays, in vitro transduction, and silver stain approaches we show that there is no difference in purity...so excited? For starters, we believe that this approach can be adapted by vector cores and laboratories...investigated 3 serotypes it’s possible that this approach will be beneficial for a host of other AAV serotypes...serotypes; it will be exciting to test this approach with different capsids. Most importantly, because .... "Substantial overproduction of antibodies by applying osmotic pressure and sodium butyrate." Biotechnology...

  48. Prime Editing: Adding Precision and Flexibility to CRISPR Editing

    Type
    Blog Post
    Published
    Jan. 13, 2025, 9:49 p.m.
    ...unedited strand (Chen et al., 2021). The team applied this approach to both the PE2 and PE3 systems, naming ...and flexibility to the CRISPR editing world. The approach, originally published in Nature, is a “search-.... The initial result is a heteroduplex, with overlapping strands of edited and unedited DNA. The heteroduplex...editing typically needs to be optimized for each application, and the efficacy varies widely — it still almost...optimized enzyme can be used with any of the PE2-5 approaches, sometimes referred to as PE2max, PE3max, etc...improved efficiency, although both enzymes still approach or exceed PEmax editing efficiencies for short...prime editing. To protect the 3′ end, the Liu lab appended RNA pseudoknots, calling this longer RNA an “engineered...

  49. Antibodies 101: Multiplex Immunofluorescence

    Type
    Blog Post
    Published
    May 23, 2023, 1:15 p.m.
    ... achieve multiplex IF. One of the most common approaches is to use sequential cycles of primary and secondary...detection with a mixture of primary antibodies and appropriate secondary antibodies. The general process is ... 1: Schematic of multiplex immunofluorescence approaches. A) Sequential Multiplex IF, where one target...multiplexed IF, many of the same considerations apply as for standard IF. However, there are a few specific...primary antibodies that have been validated in IF applications (find IHC validated or ICC validated antibodies... you have some complex patterns with several overlapping targets to interpret. Check out (Jambor et al...favorite tips and tricks, leave a comment below. Happy imaging! References and Resources References Andrews...

  50. Viral Vectors 101: Viruses as Biological Tools

    Type
    Blog Post
    Published
    March 21, 2023, 1:15 p.m.
    ...and deliver any cargo you fancy (size limits do apply, however.) Why use a virus? Viruses are essentially...low immunogenicity which makes them ideal for applications with whole organisms. Finally, viral delivery...delivery is typically straightforward across applications, while electroporation of a human, for example, isn... post. If a viral vector seems like the right approach for your experiment, you’ll need to decide exactly...retro viruses can accommodate a cargo up to approximately 8 kb, but viral packaging is less efficient ... In fact, AAV gene therapies have already been approved in the US for multiple diseases, including hemophilia... (~4.5kb From basic science to clinical applications, viruses are true bench to bedside tools. Keep...
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