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Showing: 401 - 440 of 1006 results

  1. Addgene's Tips for Plasmid Quality Control

    Type
    Blog Post
    ...most important step in ensuring quality, as it will catch any errors made during the cloning, sample handling...One of the best things about sharing plasmids through Addgene is that we provide an added level of confidence...samples coming through our doors, the first step to ensuring plasmid quality is to stay organized. Here at ..., antibiotic resistance, restriction sites used during cloning, and most importantly, the predicted full...

  2. Synthetic promoter AAVs for cell-type specific expression in retinal cells

    Type
    Blog Post
    ...retinal ganglion. Rather than using naturally occurring promoters to drive transgene expression, the Roska...one type of cell, with the remaining expression occurring in only MĂŒller glia cells, a common contamination...retina.  Find the AAV synthetic promoter plasmids! Comparing synthetic promoters performance in mouse, monkey...targeted cells in monkey and human retinas. By comparing the performance of promoters across these three...be paired for targeted expression. This same pairing of synthetic promoters were also used to target...

  3. A Look at Addgene's QC Process

    Type
    Blog Post
    ...completely different than what was originally intended. Sharing unverified plasmids can lead to far reaching consequences...standardization provides security for requestors, ensuring that the plasmid they ordered contains the correct...QC topics that may require additional attention during analysis. These include topics such as: locations... features. Most of these regions are identified during the QC process, following sequencing and assembly...30% of deposited plasmids! We are committed to ensuring that we maintain a high standard of sequence accuracy...

  4. CRISPR 101: Targeting Non-Coding RNAs with CRISPR/Cas9

    Type
    Blog Post
    ...made of protein-coding genes, and you might be wondering what the rest of our genome could possibly be ... modulate ncRNA expression without permanently altering the DNA, CRISPR interference (CRISPRi) and CRISPR...high specificity, particularly in applications requiring smaller delivery systems (Zibitt et al. 2021).... a number of different options. By carefully considering your ncRNA of interest, you should be able to...RNA-targeting CRISPR enzymes for transcriptome engineering References Jinek M, Chylinski K, Fonfara I, Hauer...

  5. 7 Simple Strategies to Resolve Conflicts with Difficult Supervisors

    Type
    Blog Post
    ...their promises, Wet-blanket negativist, who can bring down the morale of a group with their pessimism,...communicate their ideas confidently while also considering the needs of other people. Some students and ...Regardless of your supervisor’s management style, bring challenges to his or her attention as soon as you...signed, or manuscripts that need to be reviewed, bring them to the meeting. This will make it easy for ...problem by stating the facts and explain how it is interfering with your work. Avoid talking about your emotions...

  6. Transferable Skills: Negotiation

    Type
    Blog Post
    ...something done not by people wearing a lab coat, but instead by people wearing business suits discussing ...transferable skills while working in the lab and by offering advice on areas where you might need some help...you are negotiating. Then take a moment before, during, or after a negotiation to think about the situation...experience in the area I want to explore while also ensuring that my work gets completed? I want my company...

  7. Antibodies 101: Conventional vs Spectral Flow Cytometry

    Type
    Blog Post
    ...special considerations. Conventional flow cytometry During conventional flow cytometry, light emitted by a...distinguish these two fluorophores from one another during spectral unmixing.     Figure 2: The ...a three color panel or a 40 color panel! When preparing a spectral flow experiment and panel, there are...telling exactly how many Cy7 molecules are added during that particular batch. This can present particular...upon antibody binding, leading to discrepancies during unmixing. Additionally, it is critical to include...

  8. Google Forums Round Up: First Impressions of NgAgo

    Type
    Blog Post
    ...perform functional tests. The newest genome engineer sharing the stage with much-lauded CRISPR-Cas9 is DNA-guided... endonuclease NgAgo! We'll discuss how NgAgo is faring with users in a minute, but, to start, let's review...cleavage (1). Both the CRISPR-Cas9 and NgAgo genome engineering methods have their own pros and cons (2). The...protein to load an ssDNA guide. It is possible that, during translation, only a small fraction of nascent NgAgo...Development and applications of CRISPR-Cas9 for genome engineering." Cell 157.6 (2014): 1262-1278. PubMed PMID: ...

  9. PITChing MMEJ as an Alternative Route for Gene Editing

    Type
    Blog Post
    ...Introduction There are three primary methods for repairing DNA after a double-stranded break. HDR copies ...mutations seen with TALENs and CRISPR. MMEJ is active during M and early S phases, when HDR is inactive, and...Non-homologous End Joining Read About Other Genome Engineering Tools like Sleeping Beauty Mutase Find the Ideal... genome engineering... Mary Gearing...

  10. Transgenic Organisms, Cas9 Gene Drives, and Appropriate Safeguards

    Type
    Blog Post
    ...heterozygote into a homozygote and ensuring that all future offspring inherit the endonuclease gene. As...As we pointed out last year2, delivering a DNA cassette encoding the cas9 gene and single guide RNAs (...shared by everyone, he is deeply concerned with ensuring that gene drives and other collective technologies...selfish genes as tools for the control and genetic engineering of natural populations. Proc. Biol. Sci. 270,...

  11. The Effect of COVID-19 on Liu Lab Plasmid Requests From Addgene Data

    Type
    Blog Post
    ... the Addgene blog Learn more about plasmid sharing during COVID-19 Resources on Addgene.org Find plasmids...False, type='scriptV4', hide_playlist=True, viral_sharing=False, embed_button=False, autoplay=False, hidden_controls...to follow an exponential decay model, with dips during weekends and significant holidays. Figure...likelihood by the corresponding factor 𝜙z if t is during event z. The "events" we considered for our model...

  12. Summer SciComm Series: Modes of Communication

    Type
    Blog Post
    ...If you're interested in sharing your science, one of the big questions you'll be faced with is "how?"...for longer educational videos, and Twitter, for sharing updates, new deposits, and new blog posts!  ...  of proficiency.  What kind of content are you sharing?  Sometimes your subject matter will drive the ...fair, or a semester-long after school program? Comparing your desired outcome to the limitations and potential...response. High connection levels and interaction. Can bring in things to be physically interacted with. Public...

  13. AAVs in Retinal Gene Therapy

    Type
    Blog Post
    ...presence of antigens (such as viral vectors) without triggering a destructive inflammatory immune response. At...after the cloning of the RPE65 gene, a naturally occurring animal model, the RPE65−/− dog, was characterized...retinal neurons. Nowadays a toolbox of naturally occurring and engineered AAV serotypes that further expands...being done to further improve the technology: engineering of new capsid variants, designing expression ... optimize transgene levels, and large scale manufacturing of viral vectors to support trials for prevalent...

  14. Viral Vectors 101: The Retroviral Lifecycle

    Type
    Blog Post
    ...most cases, interested in transferring genes to mammalian cells (i.e. transferring DNA), yet retroviruses...producer cell membrane, and acquired as an envelope during exocytotic budding, as the viral particle emerges...transfer of RT, which would actually occur in step 2 during uncoating. After uncoating, reverse transcription... integrated by the viral integrase protein (IN) during step 3 (King et al., 2008) (Hu & Temin, 1990). ...

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

    Type
    Blog Post
    ...suppose you have a piece string that’s 10 meters long. You twist and roll the string into a tight ball. Now...in a way that brings linearly distant regions close together. gRNA Selection and Filtering for Off-target...Now the two ends of the string, instead of being 10 meters apart, are mere millimeters apart. Similarly...

  16. CRISPR Antimicrobials

    Type
    Blog Post
    ...bacteria and prevents sensitive bacteria from acquiring resistance genes. Yosef et al. suggest that it...not previously infected by the lysogenic phage, rendering the population sensitive to antibiotics. Created... Lemire, Diana P. Pires, and Timothy K. Lu. “Engineering Modular Viral Scaffolds for Targeted Bacterial...Addgene Blog CRISPR Methods for Bacterial Genome Engineering Treating Muscular Dystrophy with CRISPR Gene ... Mary Gearing...

  17. CRISPR 101: Cytosine and Adenine Base Editors

    Type
    Blog Post
    ...resulted from excision by uracil N-glycosylase (UNG) during base excision repair. Adding a second copy of the...off-target mutations than those derived from naturally occurring cytidine deaminases. Adenine base editors The ...compared to ABEmax. In an alternative approach, engineering and directed evolution of the TadA domain has...) This post was originally written by Mary Gearing in August 2016 and was updated by Jennifer Tsang...Y., Guan, Y., Siwko, S., 
 Li, D. (2023). Re-engineering the adenine deaminase TadA-8e for efficient and...applicability of base editing through protein engineering and protein delivery. Nature Communications, ...

  18. Four Base Editing Reporters to Monitor and Enrich Editing in Real-time

    Type
    Blog Post
    ...editing reporters that can help you do just that. Monitoring APOBEC and Cas9-mediated editing in real time...results in two DSBs that can be repaired by NHEJ restoring mCherry expression. Cells can then be sorted using...editing efficiency can be easily determined by comparing cells expressing only eGFP expression to cells...real-time, it relies on DSBs which is not ideal for monitoring single base edits (St. Martin et al., 2018). ...the Harris Lab circumvents the need for DSBs in monitoring on-target DNA editing efficiency of APOBEC-Cas9...

  19. New CRISPR Tools: Cas7-11 and PASTE

    Type
    Blog Post
    ...expand and exploit CRISPR, both of which we're featuring in today's blog. Cas7-11 Development and Discovery...alongside some creative (and significant!) protein engineering. They combined Prime editing with an integrase...potential applications. But they weren’t done engineering quite yet – surely, they thought, the integration...optimized the specific attachment sites via sequence engineering, enabling even more efficient integration activity...: Adding Precision and Flexibility to CRISPR Engineering CRISPR 101: RNA Editing With Cas13  CRISPR Cheat...

  20. Supporting Reproducibility with a Connected ELN

    Type
    Blog Post
    ... data. Although it may be useful to selectively bring particular items into an ELN – e.g. an image that...the integration with protocols.io, the protocol sharing repository. As illustrated in this video the integration...RSpace becomes part of a workflow that enhances the sharing and reproducibility ecosystem protocols.io makes...help with searchability, record-keeping, and data sharing, not all of them support reproducibility in the...Reproducibility Read all blog posts about scientific sharing Resources on Addgene.org Find protocols of molecular...

  21. CRISPR 101: Targeting RNA with Cas13a (C2c2)

    Type
    Blog Post
    ...pyogenes (SpCas9), is truly an exceptional genome engineering tool. It is easy to use, functional in most species... cell stress, and RNA size distribution. When comparing LwaCas13a to shRNA, they found that LwaCas13a ...vivo RNA imaging. In short, the Cas13a molecule brings a much anticipated element of diversity to the ... the CRISPR field. We feel that the culture of sharing within the CRISPR community is an essential part...scientific community. Note: Cary Valley and Mary Gearing contributed to the update of this post. References...

  22. AAV Titers: Where Do They Come From And What Do They Mean?

    Type
    Blog Post
    ...viral genome). Empty viral capsids can be formed during viral production depending on the  experimental...usually report? Addgene and other viral vector manufacturing facilities report the physical titer of a viral...to two-fold. How can we get more reliable AAV titering? For our viral service, we currently titrate AAV...different sources in the same experiment, consider retitering both AAV in your lab. The absolute titer values...Addgene, we don’t reserve lots, but if you’re reordering the same virus feel free to email us and we can...

  23. New Acoustic Reporter Genes: Ultrasound Imaging of Gene Expression

    Type
    Blog Post
    ...studying chemical engineering. Mengtong "Tom" Duan is a PhD student studying bioengineering. They are both...smaller genetic footprint, and nonlinear acoustic scattering. The best candidate for this was the Anabaena...was sufficient to saturate expression. When engineering a cell line, cotransfect the mARG cassettes 2...acoustic reporter genes for real-time in vivo monitoring of tumors and tumor-homing bacteria. Nat Biotechnol...

  24. Tips for CRISPR Gene Editing in Mice

    Type
    Blog Post
    ...software. When you are happy with your selection, ordering the sgRNA is simple and easy. Several companies...but fret not! The key to saving time and energy during your in vivo work is to screen you gRNA in vitro...in mice to the day you have your F0 generation (barring life’s little accidents and stuff ups). This is...multiple genes by CRISPR/Cas-mediated genome engineering." Cell 153.4 (2013): 910-918. PubMed PMID: 23643243...Addgene Blog Get the Basics of Mouse Genome Engineering with CRISPR Validate Your Genome Edit Learn about...

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

    Type
    Blog Post
    ...unfamiliar, scFvs are often the building blocks for engineering proteins. So let’s get acquainted with scFvs....CD3 on T cells and a tumor-specific antigen, it brings T cells to a tumor site. ScFvs can also be fused...viral vectors like AAV, which could be useful for delivering scFvs that inhibit HIV.   References and Resources...Bruccoleri RE, Haber E, Crea R (1988) Protein engineering of antibody binding sites: recovery of specific...more about antibodies  Read about another reagent sharing platform for antibodies, the Developmental Studies...

  26. The Fluorescent Vegetables in Aptamer Soup

    Type
    Blog Post
    ...proven useful in the engineering of riboswitches. Riboswitches are naturally occurring regions in the 5’ ...into the content of Addgene’s aptamer soup by exploring the development and applications of fluorescent...Warner et al., 2014); its length reduces artefacts during live cell imaging. Bunch of baby spinach, or BoBS...transcribed by RNA Polymerase III (5S, tRNA and U6) and measuring Corn fluorescence by flow cytometry, the Jaffrey...

  27. Fluorescent Proteins 101: When GFP lets you down

    Type
    Blog Post
    ... require anaerobic conditions. In experiments requiring anaerobic conditions, proteins that bind endogenous...bilirubin (Kumagai, 2013). Next to these naturally occurring co-factor binding proteins, systems wherein a ...proteins can be accurately determined in vitro by measuring the fluorescence intensity of purified protein...usually dim relative to fluorescent probes, recent engineering efforts have increased the brightness of these..., Marieke, et al. "A FRET-based biosensor for measuring Gα13 activation in single cells." PloS one 13.3...

  28. Deep Mutational Scanning with One Pot Saturation Mutagenesis

    Type
    Blog Post
    ...then used to measure the frequency of each variant during the selection process. Each variant is assigned...of MnCl2. Disadvantages of this method include favoring of certain types of mutations, difficulty controlling...then both strands of the plasmid will be nicked during the first BbvCI digest and the whole plasmid will...direction, both strands of the plasmid will be nicked during the first BbvCI digest and the whole plasmid will...Douglas M., Jason J. Stephany, and Stanley Fields. “Measuring the Activity of Protein Variants on a Large Scale...

  29. Proximity Labeling: A Powerful Tool for Protein Complex Purification and Proteomic Mapping

    Type
    Blog Post
    ...Proximity labelling relies on the labelling of neighboring proteins within a cell with biotin. Biotin, is...promiscuous biotin ligase that can efficiently label neighboring proteins The first biotin based proximity labelling...from the ligase and bind to exposed lysines on neighboring proteins. Thus to capture protein complexes, ...electron-rich amino acids such as tyrosine on neighboring proteins resulting in their biotinylation. The...The advantage of APEX is its speed, labeling neighboring proteins in minutes rather than hours like BioID...

  30. Plasmids 101: Biotinylation

    Type
    Blog Post
    ...also ideal for signal amplification during these experiments. During detection, a biotinylated reporter... biotin. Thus something in the egg white was sequestering biotin from being absorbed. In 1940 Esmond E...can be added to either terminus of a DNA sequence during a Polymerase Chain Reaction (PCR) by using a biotinylated.... Biotin can also be enzymatically incorporated during PCR using a biotin labelled dUTP. There are multiple...

  31. Multiple Plasmids at a Low Price: Kits from Addgene

    Type
    Blog Post
    ... nonprofit institution, you can order plasmids covering a wide range of fields from Addgene. What you ...4740. You can find more info on pCri in Mary Gearing’s blog post on the system Easing the Protein Purification...Seq kit has been used in microbiology studies monitoring sequence dynamics under various selective pressures... can lead to surprisingly large fitness effects during adaptive evolution of antibiotic resistance." Proceedings...2016): 895-899. Choudhury, Rajarshi, et al. "Engineering RNA endonucleases with customized sequence specificities...

  32. Using Phosphoserine to Study Protein Phosphorylation

    Type
    Blog Post
    ...post-translational modifications, identifies a staggering ~250,000 phosphorylation events in the proteomes... led to the eventual discovery of a naturally occurring phosphoserine-tRNA synthetase that serves as an...elongation factor that accepts Sep-charged tRNAs during translation. Many ‘flavors’ of Sep-OTS plasmids...pSer-containing GFP that can be used as a positive control during optimization experiments. This version of GFP will...dephosphorylate the pSer-incorporated protein, ideally bringing the phenotype assayed closer to that of the wild...

  33. How to Negotiate a Successful Lab Start Up Budget

    Type
    Blog Post
    ...How much do you need [for your lab start up]?” A hiring chair or dean will often ask a faculty candidate...community is the ultimate value proposition. By preparing a budget before hand, you’re saving the institute...turned down a faculty position because none of the offering institutes were able to provide the resources ...If the resources aren’t available, then you’re entering a win-lose situation that can ultimately set you... be afraid to walk away from a negotiation. If securing a faculty position is your goal, then a resource...

  34. Addgene is Stopping Use of X (formerly Twitter)

    Type
    Blog Post
    ..., and as of today, will stop posting, monitoring, and resharing content on X.  Addgene deeply values our..., TikTok, Facebook, and LinkedIn. We are also exploring other social media platforms with active scientific...in the scientific community. Addgene has been considering a gradual move off X as a response to major platform...

  35. Trends in CRISPR and SynBio Technologies

    Type
    Blog Post
    ...the recent Keystone Meeting "Precision Genome Engineering and Synthetic Biology". His reflections on the...scientific community. Data on CRISPR and synBio plasmid sharing In a recent review on CRISPR technology, for example...online user forums such as http://www.genome-engineering.org and http://www.egenome.org." At the Keystone...Keystone Meeting Eric presented a poster with data comparing Addgene global deposit and distribution trends...

  36. New FLIS Plasmid (April Fools 2022)

    Type
    Blog Post
    ...accosted by dozens of tiny black dots during our stay there. Yet capturing the flies is nearly impossible, ..., due to their small size and inconspicuous coloring.  FLIS plasmid Recently, the Phuels lab deposited...everywhere find and re-contain any specimens that escape during routine lab activities.  Here, we demonstrate the...

  37. CRISPR 101: Epigenetics and Editing the Epigenome

    Type
    Blog Post
    ...impossible. With the genome engineering revolution came epigenome-engineering tools - zinc finger nucleases...enhancer regions (Mendenhall et al., 2013). By comparing gene activation when enhancers were active or ...Sauka-Spengler T (2017). Genome and epigenome engineering CRISPR toolkit for probing in vivo cis-regulatory... Mary Gearing...

  38. A Control for All Seasons

    Type
    Blog Post
    ...treated identically, used to assess naturally occurring random biological variability. If one were studying...species-on-species staining can be partially addressed with stringent staining protocols and the use of isotype-specific...standard protocol, but the primary antibody is omitted during the incubation step. Samples are incubated with...will briefly touch on.  Western blotting When comparing samples in different lanes in a western blot, ...controls address the spectral overlap that occurs during multiplexing. Compensation controls are single ...

  39. Viral Vectors 101: Optogenetic Tools

    Type
    Blog Post
    ...cell-type specific. If it is something you’re considering for your experiment, note that you cannot extrapolate...hardware types, construction, and testing used for delivering your light stimulus. This can make or break your...Photoswitching Proteins (Vol. 2173, pp. 21–51). Springer US. https://doi.org/10.1007/978-1-0716-0755-8_...2019). Cortical layer–specific critical dynamics triggering perception. Science, 365(6453), eaaw5202. https...

  40. Plasmids 101: Repressible Promoters

    Type
    Blog Post
    ...bound repressor then prevents transcription from occurring, which means that transcription is now OFF.   ... ADH1 promoter pADH1m, which maintains activity during the late ethanol consumption phase of yeast culture... al. Using Binary Systems Together You may be wondering, “why is it necessary to have all 3 of the different...experiments! This post was originally written by Mary Gearing in 2018, and updated by Hannah Dotson in Dec. 2022...Introduction and expression of genes for metabolic engineering applications in Saccharomyces cerevisiae.” FEMS...
Showing: 401 - 440 of 1006 results