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  1. Deep Dive: qPCR

    Type
    Blog Post
    Published
    Sept. 29, 2022, 1:15 p.m.
    ...appropriate choice (Panina, 2018). Look for studies specifically aimed at determining the best options for...Zhang M, Hu G, Fang F. Fudenine, a C-terminal truncated rat homologue of mouse prominin, is blood glucose-... amounts of reagents in your chemistry, unless running a multiplex reaction or on a rapid cycle machine... quick way to validate your primer probe is by running your qPCR product on an agarose gel, then cutting...real-time RT-PCR. J. Immunol. Methods 283:291–306.Panina, Y., Germond, A., Masui, S. et al. Validation of...Digital Droplet PCR for AAV QuantificationPlasmid Cloning by PCR...

  2. An Addgene Summer Internship

    Type
    Blog Post
    Published
    Aug. 8, 2024, 7:22 p.m.
    ...expand my knowledge more than ever before. Before joining Addgene, I had spent most of my junior year stressed...well as complete the lab safety and cybersecurity training before stepping foot into the lab itself. I appreciated... as equals, because as soon as I had passed my training, I dove straight into my capstone project. A capstone...wrong. My capstone project was based on plasmid cloning and I, the researcher, got to choose what gene ... winner. We used the next 4-5 weeks to work on cloning the Syntaxin3 gene in a pLenti vector backbone....assembly but decided to go ahead anyway. We were running on a time crunch and knew that if we were to restart...

  3. Addgene at the ASM Microbe 2016 Conference

    Type
    Blog Post
    Published
    July 7, 2016, 2:30 p.m.
    ...up the Addgene booth on Thursday evening, I stayed to see the Opening Keynote address with Bill Gates.... lab). Bright and early on Saturday morning, John Doench’s opening presentation at the CRISPR screens ...well-optimized CRISPR screening technology. Addgene got several shout-outs for making popular screening libraries ...

  4. CRISPR Methods for Bacteria: Genome Engineering, CRISPRa, CRISPRi, Base Editing, and More

    Type
    Blog Post
    Published
    Sept. 28, 2020, noon
    ...contain BsaI sites for easy cloning of a gRNA of interest. Cytidine and adenine base editing in Streptomyces...breaks are lethal because the non-homologous end-joining (NHEJ) repair pathway is not very robust. In many...applications for which they’ve been used. The beginnings of bacterial CRISPR engineering One common way...template, using Gibson Assembly or restriction enzyme cloning. Streptomyces bacteria are more recombinogenic ...directly modify a given gene. CRISPR-tranposons By combining CRISPR editing and transposons, the Sternberg ...inducer, in this case anhydro-tetracycline (aTc). By cloning in two tandem copies of the gRNA cassette, one ...Sun lab demonstrates the utility of cytidine and adenine base editors that enable C-to-T and A-to-G base...

  5. Transferable Skills Guide: Teamwork

    Type
    Blog Post
    Published
    Nov. 28, 2017, 2 p.m.
    ...you need until the team really starts functioning (or malfunctioning). Clear lines of communication Few ...that many researchers feel that they lack formal training in a variety of transferable skills. At Addgene...The famous discoveries and experiments I’d been learning about for years were usually the products of teams...I can also orchestrate the formation of high-functioning-but-temporary teams that have a specific purpose...snapped out of the phase of my life that had me listening to Simon & Garfunkel’s “I Am A Rock” on repeat...a team is a really good way of creating and strengthening those social bonds. So never mind how much better...

  6. Scientists Map the SARS-CoV-2-Human Interaction Network

    Type
    Blog Post
    Published
    April 9, 2020, 1:15 p.m.
    ...FedEx envelopes in a single day -- all while maintaining social distance.  From front to back: David Gordon... fight against coronavirus We are just at the beginning of understanding how SARS-CoV-2 interacts with...used HEK293T cells in our first study, but we're planning to soon expand to other cell types, including ...community's help. You could be the next scientist joining in this worldwide fight against SARS-CoV-2. As ...flanked by EcoRI and BamHI restriction sites - so cloning projects should be fun and straight-forward. All...confirmed expression via mass spec for two of the remaining viral proteins. Only Nsp11 ran bigger than expected...

  7. Trading Spaces - Visiting Scientist Edition

    Type
    Blog Post
    Published
    Aug. 22, 2023, 1 p.m.
    ...If you’re interested in learning a new skill, getting access to equipment you don’t have, or maybe both...researcher remains employed by their home institute, meaning the host university does not hire or pay them (...current institute. A common reason is hands-on training in a technique that only a few labs have expertise...this expertise will provide you with guidance and training so that you yourself can become an expert and ... Identify reagents you will need, any required training, and a priority list of experiments you hope to... Visiting scientist positions take commitment, planning, and funding – they may not be right or necessary...

  8. Hot Plasmids - June 2019 - Optogenetics, Acoustic Reporter Genes, microRNAs, and the CRISPR-Cas9 system CHIME

    Type
    Blog Post
    Published
    June 6, 2019, 12:41 p.m.
    ...PMID: 30704911. Acoustic reporter genes for noninvasive imaging of microbes in mammalian hosts Article...podcast segment In recent years, scientists are beginning to find connections between the microbiome and...largest miRNA sensor collection reported to date, containing all 620 sequences of probable mature human miRNAs...consists of a library of reporter plasmids, each containing two fluorescent proteins and four repeats of ...plasmids is amenable to Golden Gate assembly for turning the single-input reporters into multi-input cell...al., Nat Commun, 2019. PubMed PMID: 29934631. Screening immune cell lineages in vivo using the CRISPR-...

  9. Sweating the Small Stuff: Details in the Lab

    Type
    Blog Post
    Published
    Feb. 8, 2022, 2:15 p.m.
    ... all scientists? Curiosity? Passion? A love of learning? No. Or at least not entirely. What truly unites...the Issue You can’t fix an experiment by simply running it unchanged over and over again, and the same ...works, so do it anyway.  You may find that after running a protocol a few times, you no longer need the ...few key reminders on a sticky note   Visual Planning  Plate maps, pre-labeling tubes, and even drawing...stressed, or rushing. I would spend the time quietly running through the experiment in my head, double-checking...another short "quiet period" instead of immediately running to the next thing.  It may seem counterintuitive...

  10. Plasmids 101: Screens vs. Selections

    Type
    Blog Post
    Published
    Nov. 10, 2022, 2:15 p.m.
    ...component. Alternative screening techniques While CRISPR and drugs dominate the screening scene, there are ... a selection, you might put cells into a drug-containing media in which only cells which can tolerate ...is typically not feasible to test in any given screening experiment. Thus, a limited panel is usually selected... sensitive enough? No one wants to spend time screening hits that aren’t “real” or novel for their purposes...Dijkhuizen, L. Directed evolution of enzymes: Library screening strategies. IUBMB Life, 61: 222-228 (2009). doi...

  11. Custom CRISPR Screens & the Green Listed Software

    Type
    Blog Post
    Published
    July 11, 2017, 2:30 p.m.
    ...sequences for cloning purposes.  Press run and wait for the generation of an output folder containing several...contain 4-12 gRNAs/gene, which results in libraries containing up to around 200,000 different gRNAs. It has ...several files. These include: Output: A text file containing full information about the suggested gRNAs to...generated. Output_Short: A condensed text file containing a full and a short name for each gRNA, as well... vectors and genome-wide libraries for CRISPR screening." Nature methods 11.8 (2014): 783-784. PubMed ...Addgene.org Learn More About CRISPR Find CRISPR Screening Libraries Browse All CRISPR Plasmids ...

  12. Cancer and the Immune System: Deciphering the Relationship

    Type
    Blog Post
    Published
    March 14, 2017, 2:30 p.m.
    ...knowledge with our bodies’ natural abilities, we’re gaining new ground in the fight against cancer in its many...between tumors and the immune system We’ll begin by learning how the immune system naturally interacts with...concept of immunoediting, by way of favorably attaining the equilibrium stage. This can be called “stalemate...and open questions in cancer treatment We are beginning to see the fruits of many years of labor with ...enter the clinic, physicians and researchers are learning much about how treatment regimens can be improved...effective. Some important factors researchers are beginning to focus on include: Cell heterogeneity Tumors...Collection Pages Plasmid Grant to Aid Cervical Cancer Screening in Ghana Modulate Cancer Signalling Resources...

  13. Celebrating 15 Years of Scientific Sharing

    Type
    Blog Post
    Published
    Jan. 14, 2019, 2:08 p.m.
    ...At Addgene we will never ever stop planning, perfecting, and learning, but there are a lot of things we...support more new projects. Diversification is happening. The Addgene team will have some fun challenges...external website appearance and functionality are happening and this strengthens our image in the world. ...inclusive workplace for everyone. We had Unbiasing Training in place before it was cool or a necessary result...research  Learn about our viral service Check out job openings at Addgene  ...

  14. Antibodies 101: Polyclonal Antibodies

    Type
    Blog Post
    Published
    July 14, 2021, 1:15 p.m.
    ...method. These proteins bind the IgG heavy chain. Running the serum over a column of resin crosslinked to... In this case, the serum is run over a column containing the target protein, and any antibody recognizing...pre-adsorb the antibodies, pass them through a column containing immobilized serum protein from other species....target protein. While this can increase background staining (see next section), it may be advantageous if ...might experience variability in the strength of staining and background noise. If you do use different ...choose the appropriate controls to be sure the staining you are seeing is real. It’s important to include...

  15. Overcoming the Challenges of Lentiviral Production

    Type
    Blog Post
    Published
    Aug. 7, 2018, 12:31 p.m.
    ...trying to produce virus of a usable titer, or fine-tuning selection and expression in your target cell line...that usually encodes VSV-G, a packaging plasmid containing structural genes and a transfer plasmid carrying...will also affect experiment optimization. Before beginning an experiment, it is recommended that you try ...challenging. With these considerations in mind, when planning an experiment users should weigh the benefits ...collected, filtered, and titered. A major obstacle in obtaining usable lentivirus is producing a high enough concentration..., at Addgene we typically concentrate 100-fold meaning that a 10 cm2 dish preparation of virus will only...

  16. Popular Retroviral Vectors and Their Uses in Scientific Research

    Type
    Blog Post
    Published
    July 24, 2018, 12:51 p.m.
    ...Retroviral vector for cloning and expressing your gene of interest with GFP expression.  Cloning and expressing...CRISPR pooled libraries that allow functional screening of thousands of genes in genome-wide experiments...regulation, gene of interest expression, knockout, and screening. There are many more useful viral vectors available...collection can be used for in vivo phenotypic screening of small-molecule libraries and can be combined...lentiviral expression vectors (empty backbones) here. Cloning and expressing new shRNA sequences pLKO (Plasmid...

  17. Plasmids 101: FLEx Vectors

    Type
    Blog Post
    Published
    April 28, 2016, 2:30 p.m.
    ...off expression of one gene, while simultaneously turning on the expression of another! What Are FLEx switches...FLEx switch that turns BFP expression off, while turning on mCherry expression. For this FLEx switch to ...the first recombination step will invert the intervening DNA fragment using either loxP or lox511 sites...FLEx systems are generally considered "Cre-On" meaning that your gene of interest starts in the inverse... genOway, we are able to distribute materials containing FLEx technology. Read the genOway press release... Michelle Cronin...

  18. Mobile-CRISPRi: Bringing CRISPRi to Diverse Bacteria

    Type
    Blog Post
    Published
    April 4, 2019, 12:53 p.m.
    ...for genome-wide CRISPRi library screening experiments where maintaining library diversity is important,...-CRISPRi into bacteria. Top: A Tn7 transposon containing the CRISPRi components and a plasmid with the...integrated copy of CRISPRi. Bottom: An ICE element containing the CRISPRi components is transferred to recipient...essential genes were less represented while bacteria containing guides targeting non-essential genes remained...Kyoung-Hee, et al. "A Tn7-based broad-range bacterial cloning and expression system." Nature methods 2.6 (2005...

  19. No Llamas Required - Synthetic Nanobodies Against Membrane Proteins

    Type
    Blog Post
    Published
    June 18, 2020, 1:15 p.m.
    ...References Geertsma ER (2013) FX Cloning: A Versatile High-Throughput Cloning System for Characterization ...SB_convex. These scaffolds contain serines and threonines at positions that can be randomized for creating...protein (sybody), ribosome, and mRNA. During the screening process, displayed sybodies that bind to the biotinylated...pDX_init plasmid, using the fragment exchange (FX) cloning technique which uses Type IIS restriction sites... phage display screens are subcloned (using FX cloning) into the expression vector pSb-Init, which attaches...

  20. Transferable Skills: Negotiation

    Type
    Blog Post
    Published
    March 3, 2022, 2 p.m.
    ...found that many researchers feel they lack formal training in a variety of transferable skills. At Addgene... to be done (Who is responsible for making the running buffer this week?). Scientists negotiate all the... on this transferable skill without any formal training or coursework. First, you have to develop an awareness...settings, creative negotiation skills are key to maintaining and growing relationships with your colleagues... them to ensure we create a win-win situation? Learning about and respecting cultural differences is a... academic committees as a tenured professor or signing contracts when working for a business development...

  21. Polymerase Chain Reaction Overview and Applications

    Type
    Blog Post
    Published
    Aug. 10, 2021, 1:15 p.m.
    ...multitude of scientific techniques such as molecular cloning or molecular diagnostics.       Steps of PCR...End point PCR is most commonly used in molecular cloning, sequencing, and genotyping. It’s extremely useful...of DNA. This amplicon can be used in molecular cloning to create a plasmid of interest, for detecting ...PCR Amplify a region of interest  Molecular Cloning Site-directed mutagenesis Molecular Diagnostics...Droplet Digital PCR for AAV Quantification Plasmid cloning by PCR  Plasmids 101: Colony PCR Site Directed ... primer  Molecular Biology Reference Molecular Cloning Techniques ...

  22. Production of Virus in Insect Versus Mammalian Cells

    Type
    Blog Post
    Published
    Nov. 5, 2024, 8:32 p.m.
    ... can be produced in many different cell types, spanning both prokaryotic and eukaryotic organisms. Typically... requires three plasmids — a transfer plasmid containing your gene of interest plus two packaging plasmids... available, so you’ll only have to worry about cloning your transfer plasmid. The transfer plasmids have...media every 12 to 24 hours. Processing the virus-containing supernatant will depend on the virus type. AAV... be produced in insect cells, but they require cloning specific AAV or lentiviral machinery into a baculovirus...bacmids are readily available, so the amount of cloning required is about the same between insect and mammalian...

  23. Tips for Using BLAST to Verify Plasmids

    Type
    Blog Post
    Published
    May 29, 2014, 1:29 p.m.
    ...sequence into the Subject Sequence box that appears. Aligning two nucleotide sequences is probably the fastest...reactions are imperfect and sequence near the beginning or end of a reaction is often unreliable, we suggest...such as when you attempt to verify the starting methionine of a gene but the blastx results start the alignment... not filter low complexity regions by default, meaning that you do not have to remember to adjust any ...nucleotide or protein sequences. We also use COBALT for aligning multiple protein sequences, particularly for comparing... to help other labs speed up their plasmid and cloning verification steps and free up more time for using...

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

    Type
    Blog Post
    Published
    Jan. 13, 2025, 9:49 p.m.
    ...relatively low, meaning that most Cas9-induced DSBs are repaired by non-homologous end joining. As a result...double nicking increases indel formation slightly. Designing the sgRNA with a spacer that only binds the edited...mismatch repair contributed to a large number of unintended prime editing outcomes, including indels. Therefore...in unpredictable ways. In some cases, however, combining an evolved RT domain from PE6a-d with an evolved...binding, but pegRNAs have an extended 3′ tail containing the reverse transcriptase template and primer...efficiencies compared to PEmax with normal pegRNAs, but combining PE7 with epegRNAs resulted in similar or reduced...

  25. Transferable Skills Guide: Identifying Your Transferable Skills

    Type
    Blog Post
    Published
    Jan. 11, 2018, 3:29 p.m.
    ...Solving Creativity Data analysis Listening Negotiation Career Planning Resources Cross team communication...until it is identified by another person. Similar reasoning may also apply to other acquired skills.” It is... we are, but there is also value in knowing and owning your transferrable skills yourself so that you ...strengths and weaknesses, and view everything as a learning experience. If your first reaction to an opportunity...didn't even know you possessed. Think about your shining moments. Recall a few times you really felt great...

  26. The Importance of a Fun Workplace: Company Culture at Addgene

    Type
    Blog Post
    Published
    Sept. 3, 2015, 2:30 p.m.
    ...tirelessly to get Addgene from a concept to an up and running repository. As they built the foundation for what..., our founders and early employees spent this remaining time by elaborately pranking each other, dressing...any have some fun together! Laura’s Top 10 Event Planning Tips! 1. Take suggestions Some of Addgene’s best...low participation numbers for an event you are planning, dangle the carrot (or more popularly- cookies...people know exactly where to be and when. This pre-planning will avoid chaos and delays in getting the fun... sure people know that there is something fun happening! Addgene actually has chimes outside of our break...

  27. New Tools Enable CRISPRa for Neuroscience Applications

    Type
    Blog Post
    Published
    July 2, 2019, 12:51 p.m.
    ...through experience. However, studying the genetic underpinnings of these processes has been complicated by the...-optimized viral vectors, and high-throughput screening approaches. Here, we’ll highlight these recent...lentiviral vectors can be found here. High-throughput screening strategies Although the tools described above ...Lab, which developed a subgenome-scale CRISPRa screening approach in embryonic stem (ES) cells to determine...from undifferentiated cells at the end of the screening process. However, while the neuronal marker TUJ1...gymnastics for sensitive applications such as CRISPR screening. The plasmids used by the Qi Lab for this particular...

  28. Overcoming the AAV Size Limitation for CRISPR Delivery

    Type
    Blog Post
    Published
    Sept. 16, 2020, 1 p.m.
    ...genes in mouse muscle and eye tissue. Neisseria meningitidis Cas9 (NmeCas9) at 1,082 amino acids, can also...catalytic domain of RNA deaminase ADAR2. Constructs containing the ADAR2 truncation ADAR2DD(delta984-1090) are...virus delivery and genome editing by Neisseria meningitidis Cas9 in vivo. Genome Biol 19: . https://doi.... Comander J, Liu Q, Liu DR (2020) Cytosine and adenine base editing of the brain, liver, retina, heart...Kriz AJ, Zetsche B, Shalem O, Wu X, Makarova KS, Koonin EV, Sharp PA, Zhang F (2015) In vivo genome editing... Schmid-Burgk J, Zetsche B, Gao L, Makarova KS, Koonin EV, Zhang F (2019) Engineering of CRISPR-Cas12b...

  29. Mesothelioma - Causes, Symptoms, and Treatment

    Type
    Blog Post
    Published
    Dec. 13, 2016, 3:30 p.m.
    ...that can be used to treat it. For those who are running out of hope with more traditional treatments, such...mesothelioma is a form of cancer that develops in the linings of certain areas of the body, including the lung...less frequently, ingested), it can get into the lining of the lungs or travel to one of the other areas... a result, mesothelioma is often misdiagnosed, meaning that by the time it has caught it has usually started...mesothelioma – that is, mesothelioma that forms in the lining of the abdomen (peritoneum) – have the option of...Resistance Plasmid Grant to Aid Cervical Cancer Screening in Ghana Additional Resources on Addgene.org ...

  30. Antibodies 101: The Four ELISAs and When to Use Them

    Type
    Blog Post
    Published
    July 25, 2024, 1:15 p.m.
    ...generally have higher levels of background staining. Antigen-containing samples often include undesirable molecules...non-specifically to assay components and increase background staining. Direct ELISAs also lack the signal amplification...a capture antibody and then add their antigen-containing sample. The target antigen binds to the capture...antigen and then they preincubate the antigen-containing sample with their conjugated primary antibody...cross-reactivity Higher level of background stainingLess sensitivityLess specificityLimited availability...

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

    Type
    Blog Post
    Published
    Oct. 27, 2015, 3 p.m.
    ...Observing a bright signal for the first time after cloning an FP fusion with your favorite protein of interest... an FP within a fusion construct [1,2], i.e. positioning of the FP sequence relative to cell compartment...C-terminus [3]. You must therefore determine your cloning strategy and decide whether you will create an ... experimental needs. Equally important, before cloning you must consider whether and how well your FP ...fluorescing (see Figure 2) [5].  When using FPs containing two cysteine residues and a sequence intended... al. "Protein oligomerization modulates raft partitioning and apical sorting of GPI-anchored proteins....

  32. Important Considerations in Optogenetics Behavioral Experiments

    Type
    Blog Post
    Published
    Oct. 1, 2015, 2:30 p.m.
    ...might be contextual fear conditioning (reviewed in [4]). Contextual fear conditioning is a behavioral experiment...prolonged inhibition of CA1 neurons attenuated conditioning, which suggests that other regions of the brain...technique in neuroscience it has a bit of a steep learning curve and many opportunities for mistakes. The...University.  His interests include the role of learning and memory in opioid addiction and science communication...Kheirbek, Mazen A., et al. "Differential control of learning and anxiety along the dorsoventral axis of the...

  33. Replacing Paper: Tips for Choosing an Electronic lab Notebook

    Type
    Blog Post
    Published
    July 10, 2018, 1:08 p.m.
    ...schedules more organized. From the alarm in the morning, to the daily news briefing, to quick notes, to...users with a holistic means to manage their work, combining data, team, inventory and project management functionalities...Implementing an electronic lab notebook starts with defining your lab’s needs The values of ELNs show in the...your lab members will use the ELN. If your lab is planning to grow in the upcoming years, it is also good...include comments, notifications, sharing files, assigning team members to certain tasks, etc. How is security...Product Marketing Manager at SciNote, interested in combining scientific research with tech advances. You an...

  34. Adeno-associated Viruses (AAVs) for Genome Editing

    Type
    Blog Post
    Published
    March 27, 2018, 1:32 p.m.
    ...vectors for your specific gene, the lab recommends designing homology arms with the modification of interest...synthesis company such as IDT or Genscript, and cloning them sequentially into the chosen acceptor vector...that causes Hemophilia B. Unfortunately, work combining CRISPR with AAVs for improved genome editing is...gene fusions. The current state-of-the-art in combining the AAV and CRISPR-based approaches is described... Medicine. His research currently focuses on determining the mechanisms and consequences of cohesin gene...Epstein, Benjamin E., and David V. Schaffer. "Combining engineered nucleases with adeno-associated viral...

  35. Antibodies 101: Introduction to Immunofluorescence

    Type
    Blog Post
    Published
    Nov. 16, 2021, 2:15 p.m.
    ...the most important considerations to make when planning and performing an IF experiment. What is immunofluorescence...include: permeabilization, antigen retrieval, and sectioning. Blocking Block is used to reduce non-specific...experimental goals. Example steps include: counterstaining, mounting sample on slides, and confocal   ...your most pressing research questions, but also stunning images (am I the only one with IF results as artwork...    References and resources References Im K, Mareninov S, Diaz MFP, Yong WH (2018) An Introduction to...to Performing Immunofluorescence Staining. In: Methods in Molecular Biology. Springer New York, pp 299...

  36. An Integrin Antibody Toolkit from IPI

    Type
    Blog Post
    Published
    March 28, 2024, 1:15 p.m.
    ...short intracellular ends and large ectodomains containing ligand-binding heads and long legs that protrude...affinity. This extended-open state is key to strengthening ligand binding, mediating cell adhesion and ...technology, are directed to integrins that bind the Arginine-Glycine-Aspartate (RGD) amino acid sequence in...displayed by a subset of the RGD-binding integrins containing the αV subunit. Using these affinity reagents...education in protein science, IPI hosts events and training courses on technologies and laboratory techniques... website and stay informed on future events by signing up for the IPI newsletter. The collection of 26...

  37. How to Negotiate a Successful Lab Start Up Budget

    Type
    Blog Post
    Published
    Sept. 1, 2015, 2:30 p.m.
    ...start defining keywords you’ll want to find in candidates’ resumes or CVs; “vivarium”, “weaning”, “colony...developing a budget, I suggest that you focus on defining these resources and not on the financial aspects...your current P.I., lab manager, or administrator containing all of the materials that you’ve used within ...colony”, etc. This will help save time by screening out irrelevant candidates, and, more importantly, it...Channel your inner curiosity and use your research training to help you find other solutions to reach your...

  38. Synthetic Biology, Artificial Chromosomes, iGEM & More - Interview with Tom Ellis

    Type
    Blog Post
    Published
    Nov. 29, 2017, 2 p.m.
    ...advice for anyone pursuing a career in academia. Learning how to build a lab and a research program and ... biology to the UK with foundational research, training, startups, and real applications of SynBio. Chapter...seen with things like DNA synthesis, Golden Gate cloning, and CRISPR. The yeast synthetic genome project... of Cambridge before spending time in biotech screening direct interactions between drugs and promoters...Nonprofit BioBuilder Plasmids 101: Golden Gate Cloning Learn About Working at a SynBio Startup in Our ...

  39. Microbes: Look, Listen, and Tell

    Type
    Blog Post
    Published
    Aug. 28, 2018, 12:04 p.m.
    ...may think about sitting amongst a sea of chairs listening to talks all day. But nope, not at the American...everyone - everywhere! From ‘looking at’ to ‘listening to’ microbes But seeing microbes through a microscope...Shapiro from Caltech presented an exciting way of listening to microbes by using non-invasive methods for ...approaches to presenting the microbial world After learning about using sound to visualize bacteria in the...Raymond W., et al. "Acoustic reporter genes for noninvasive imaging of microorganisms in mammalian hosts....

  40. How to Design Your gRNA for CRISPR Genome Editing

    Type
    Blog Post
    Published
    Sept. 24, 2020, 1:15 p.m.
    ... involves the need for single cell cloning and subsequent screening for successful edits. This is a very...important to remember that the same is true when designing gRNAs for using CRISPR technology – the “best”...and sequence are important considerations for designing your gRNAs. For indels, it's not so important ..., the error-prone nature of non-homologous end joining (NHEJ) often leads to the generation of indels ...requires not one but two rounds of single cell cloning – as a control, one should revert the edit back...

  41. Generating Mouse Models Using CRISPR/Cas9

    Type
    Blog Post
    Published
    July 12, 2016, 2:30 p.m.
    ...Many thanks to our guest bloggers, Wenning Qin and Haoyi Wang! Wenning Qin is a molecular biologist with.... This post was contributed by guest bloggers, Wenning Qin and Haoyi Wang. CRISPR/Cas9 is revolutionizing...al., 2013). Like everyone else in the world, I (Wenning) was fascinated by CRISPR and rushed to test it... hard. I was mentally prepared to go through a learning curve that would result in success only after ...broken ends together through non-homologous end joining (NHEJ), this will leave behind a “scar”, with nucleotides...

  42. What's New in CRISPR - December 2019

    Type
    Blog Post
    Published
    Dec. 17, 2019, 2:37 p.m.
    ...Synthetic Biology Find the CRISPRi toolkit SECURE adenine base editor Earlier this year, Keith Joung’s lab...to rat APOBEC1. Now, they’ve engineered SECURE adenine base editor variants using a protein-truncation...pyogenes Cas9. They called this editor Target-ACE (adenine and cytosine editor). Target-ACE exhibits increased...inactivated RNA-targeting Cas13 (dCas13) with the adenine deaminase domain of ADAR2. Now, they’ve further...this editor for C-to-U editing by evolving the adenine deaminase domain of ADAR2 into a cytidine deaminase...

  43. Modulate the Activity of 17 Signaling Pathways with One Kit!

    Type
    Blog Post
    Published
    March 22, 2016, 2:30 p.m.
    ...colleagues at Duke University, in collaboration with “cloning guru” Kathleen Ottina and David Sabatini at the...which is now available at Addgene. “We called it cloning by mail,” Wood says, and it took them about six...and his colleagues developed the kit for use in screening for pathways that drive resistance to drugs in...Tracing Read How CRISPR Can Be Used in Genome-Wide Screening Browse 28 Hot Plasmid Technologies from 2015 ... cancer screening...

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

    Type
    Blog Post
    Published
    Jan. 16, 2024, 6:13 p.m.
    ... G., Haining, W. N., & Sharpe, A. H. (2019). A CRISPR-Cas9 delivery system for in vivo screening of genes...hematopoietic stem cells with a lentiviral vector containing a gRNA. These transduced stem cells can be implanted...Huang, A. Y., Anekal, P. V., Montero Llopis, P., Haining, W. N., Doench, J. G., & Sharpe, A. H. (2024). ..., Gaudiano, E.F., Al Abosy, R., Freeman, G.J., Haining, W.N., & Sharpe, A.H. (2019). PTPN2 regulates the...

  45. Easing the Protein Purification Process with pCri

    Type
    Blog Post
    Published
    June 19, 2015, 3:08 p.m.
    ...to improve protein stability and solubility. Determining the best way to express one’s protein of interest...specialists recommend doing some research, notably determining the subcellular localization and the number of...plasmids uses the same restriction enzymes for cloning (see schematic below), enabling a researcher to... in E. coli, the use of a thioredoxin A (TRX)-containing vector can improve disulfide bond formation. ...tags Resources on Addgene.org Find Additional Cloning Kits ...

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

    Type
    Blog Post
    Published
    Feb. 18, 2015, 3:09 p.m.
    ...finger nucleases to harness non-homologous end joining repair (NHEJ) [1]. Our initial work was with TALENs...haploinsufficiency. Subsequent modifications at the remaining allele within the deletion boundaries may be produced... paired DSBs. This method allows for deletion screening by conventional PCR. Biallelically disrupted cells...transfected cells via FACS, primer validation, and screening by PCR for biallelic deletion clones:    ...paper describes their methods for CRISPR design, cloning, and delivery, as well as their techniques for ...

  47. Finding Your Science Policy Path

    Type
    Blog Post
    Published
    Sept. 22, 2020, 1:15 p.m.
    ...field from a more traditional policy background, learning the science part on the job. Each path to science...implementing the policy within the jurisdiction of the governing body. This type of work can happen at the national... professional societies, and industry through convening activities, formal lobbying, or strategy setting...The “little p” policy is non-governmental work happening at large organizations who have to develop policies... do you do science policy? Many science policy training programs and fellowships have a strong focus on...nation’s largest integrated healthcare system? Defining your why is what will make you a competitive candidate...

  48. A History of Genome Engineering in Popular Culture

    Type
    Blog Post
    Published
    Feb. 25, 2020, 2:15 p.m.
    ...simulations and machine learning to develop a predictive understanding of the adenine base editors which can... regulating and propagating life, there was no turning back. While the world of molecular genetics was...This is typically followed by non-homologous end-joining that results in gene knockouts.   Star Trek: Into...locations in the genome. This was soon followed by the adenine base editor (ABE) enzyme in 2017. ABEs rely on ...editor proteins. She is particularly interested in combining the realms of art and science. Follow her at @...

  49. A Guide to Getting Started in Undergrad Research

    Type
    Blog Post
    Published
    June 8, 2021, 1:15 p.m.
    ...Getting more scientific and professional training after earning a doctorate Considered a "stepping stone...also more than eager to help you along your path. Learning about their career paths and goals helped me reflect...research opportunities are also fantastic for broadening your experiences! These applications can be highly...years. But college, and science, are places for learning and growth! As you learn more about yourself and...EleanorWang.     References and resources References Canning EA, LaCosse J, Kroeper KM, Murphy MC (2019) Feeling...

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

    Type
    Blog Post
    Published
    March 7, 2017, 3:30 p.m.
    ...randomly chose one of my stable pools. Viruses containing BRAF gRNA (BRDN0000561167 and BRDN0001146266)... 4). The most difficult part of the assay was designing primers flanking my edits in genomic DNA and doing...gRNA-transduced samples (data not shown). Pro-Tips: 1.Designing primers against genomic DNA to amplify the gRNA... vectors and genome-wide libraries for CRISPR screening. Sanjana NE, Shalem O, Zhang F. Nat Methods. 2014...Additional Resources on the Addgene Blog Genome-wide Screening Using CRISPR/Cas9 Deliver Multiple gRNAs at Once...Lentivirus Production Websites Primer3: for Designing Primers UCSC Genome Browser: For eploring genomic...
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