...passed down through generations (Figure 1). These so called “plasmid addiction systems” require dividing cells..., plasmid addiction systems have been used as biological tools for a wide range of applications in molecular...become a powerful tool to address a diverse set of biological inquiries. References Ogura, Teru, and Sota...vaccine strain CVD 103-HgR." FEMS Immunology & Medical Microbiology 57.1 (2009): 69-79. PubMed PMID: 19678844...

... ligands (Figure 2). Biosensors, which include calcium and voltage sensors, are the broadest category ...catalog If you’ve been following our blog, you may recall our first 20th anniversary story on CRISPR, where... vectors available in the repository. GCaMP, a calcium indicator (biosensor, by Addgene’s categorization...Waxmonsky, N. C., Swanson, L. E., & Fan, M. (2016). Practical Considerations for Using Pooled Lentiviral CRISPR...

...contamination. For those labs that prefer a more economical mode of detection, Uphoff et. al. outline a do...engineered with non-native envelopes, a process called pseudotyping. Lentiviruses pseudotyped with the...virus. To circumvent this problem, transductions typically employ the positively charged polycation, polybrene...lentiviral and retroviral vectors.” Human Gene Therapy Clinical Development 25.3 (2014). 175-85. Pubmed PMID: ...

...on DNA, or are enzymatically active. For this reason, recombinant proteins are typically expressed in E...medium. Induction timing is important, as you typically want to make sure your cells have first reached...your expression conditions by setting up a small-scale expression experiment to test variables such as ...

...control over where mutations are introduced, and scalability (see Supplementary Table 1 of Wrenbeck et al)...stretches of sequence could be used. However, a practical limitation is sequencing depth and cost. You need...Measuring the Activity of Protein Variants on a Large Scale Using Deep Mutational Scanning.” Nature Protocols...scanning: assessing protein function on a massive scale." Trends in Biotechnology 29.9 (2011): 435-42. PubMed...

...ICC or a tissue in the case of IHC. IF is used specifically when we visualize the antibodies using fluorescence...counterstaining, mounting sample on slides, and confocal   Figure 2: High-level overview of an ...controls to show that your antibodies are binding specifically. For positive controls, aim to use samples that...in an organism that is underrepresented in the biomedical research community. You may have to take some...

... regions of homology at their ends, which are typically created by PCR. Then, the fragments are incubated...competent cells. The success rate of this reaction is typically fairly high, so there is usually no need to screen...the bacterial chromosome (up to 100 kb) were specifically cut out via CRISPR and then assembled into a...amplify the fragments of the chromosome, which was technically challenging. Other homology based technologies...

...simple in C. elegans and bacteria. But CRISPRing typical mouse models, including creating Cre-dependent ...on both sides and the T7 promoter on one end. Typically, Easi-CRISPR works best with insertions of 2 kb...models varies from 8.5-100% of pups born, with typical efficiencies of 30-60%. Quadros et al. attribute...2.5x more animals than i-GONAD and is a more technically difficult process. Overall, the main drawback...

...desired location of edit; efficiency decreases dramatically when the cut site is >30nt from the proximal... constraints are even more exquisite for the so-called base editor Cas9, which makes DNA changes in the...nuclease-mediated off-target effects.” Further, large-scale datasets of hundreds of genetic screens using genome-wide... sequences targeting a gene to score in order to call that gene a hit.  This is an exciting time for functional...

...facilitates the integration of the transposon specifically at ‘TTAA’ sites randomly dispersed in the genome...re-integration from the piggyBac™ donor, which is typically found with the wild-type piggyBac™ transposase..., including making transgenic cell lines and genetically-modified cell lines. Furthermore, the use of ...cell engineering team.     Mathew Goodwin is a Technical Sales Specialist at Transposagen. As a gene editing...

...the details. Chapter 3 - Answering interesting biological questions using microscopy. Chpater 4 - Research...Amsterdam). He develops, characterizes and uses genetically encoded fluorescent probes. You can follow him...Plasmids for FRET Find Plasmids for Subcellular Localization ...

... BioRender.com.   Cas9 has two major lobes, called NUC (for nuclease) and REC (for recognition). The...recognizing the PAM on DNA, the CTD is also sometimes called the PAM-interacting (PI) domain. The guide RNA ...conformational state of the HNH domain also allosterically controls RuvC activity through a hinge region...Visualization, and Informatics at the University of California, San Francisco, with support from National Institutes...

...African American woman whose cervical cancer cells became a mainstain of biological study without her or her...fossil rocks and their importance in understanding biological history. 4. Please Don't Kill My Hive Beautifully...

...important immune cell type, in more complex and physiological microenvironments. These screens have primarily...molecular identifiers, which can improve the statistical power of in vivo screens. Figure 1: Using...Fellow in Arlene Sharpe’s laboratory at Harvard Medical School and is interested in improving CD8+ T cell...

...alternatives for some of the harmful chemicals in your lab. These chemicals are not only better for the environment... resources about this, and even has a list of biological samples that are stored long term at -70 °C. ...

...plasmids available at Addgene. We will still periodically focus on specific CRISPR plasmid tools more ... Feng Zhang’s lab recently developed a platform called SHERLOCK (specific high-sensitivity enzymatic reporter...will help researchers map transcription factor localization and binding via ChiP-seq. Learn more about...

...papers detailing Nobel Prize-winning research typically weren’t among the most cited. And, as scientific...online, surely times have changed. Altmetrics.org calls to expand our view of what impact looks like and... aren’t enough. Scientists are sharing what they call “raw science” – data sets, code, and, I think it...

...protein present on the virion surface – making it critical for viral attachment and fusion. Once inside a...glycoprotein’s role in infection also makes it a critical component of vaccines and the target of neutralizing...established Ebola Genome Portal at the University of California, Santa Cruz, offers sequence data on five strains...

...multiple timepoints. When the target of interest is critical to cell survival or proliferation, an additional...a flow cytometry experiment. But validation is critical to ensuring accurate interpretation of your results...to start with a screening procedure in easily genetically modified cell types (top). The specific approach..., A. M., McPherson, P., & Laflamme, C. (2023). Scaling of an antibody validation procedure enables quantification...

...few years ago, researchers introduced what’s been called a major breakthrough in cloning technology with...cassettes onto a single T-DNA in subsequent rounds, dramatically reducing the time required to generate multi-construct...multi-construct transgenic plants. In the most practical terms, what GreenGate can do is “save you half a...

...promoter. It can be used to drive Cre expression specifically in neurons. AAV Delivered Cre - A useful tool...Indeed, this AAV provides you with a means to specifically activate expression of any Cre-dependent gene...Additionally, you could use the retrograde form to specifically express Cre in neurons with axons that extend...

...sequence in live cells. This technique uses catalytically inactive CRISPR endonuclease (dCas9) fused to...scientists. You can find all of our August posts in chronological order here: Site Directed Mutagenesis by PCR...Single Base Editing with CRISPR Cas9 Activators: A Practical Guide 10 Steps to a Perfect Science Talk Developing...

...meet interesting people doing interesting things. I call this building relationships because networking has...or a particular position at their company, do NOT call this an Informational Interview. If you do this,... if it is not possible consider a phone or video call. If you do decide to use Skype, make sure to follow...

...Science and Technology Policy issued a memorandum calling on Federal agencies with over $100 million in annual...promote open science, ensure good stewardship of clinical trial data, serve society and patients, and respect...patents in a case against Myriad, an organization called Free the Data launched a public database to gather...

...used a GFP derivative to conduct subcellular localization studies or make a reporter construct. Fluorescent... mammalian cells, and it may find wide use in biological research. Photoconvertible fluorescent proteins... new transgenic line could avoid problems in localization that have been encountered by some investigators...

...research, notably determining the subcellular localization and the number of disulfide bonds in your protein...protein expression in systems other than E. coli, specifically the bacterium Bacillus subtilis and the yeast...may improve expression of proteins that natively localize there. pCri is both versatile and easy to use,...

...Institute partners with companies to provide technological resources for those studying rare disease. This...will use fluorescent proteins to monitor the localization of these genes, CRISPR to disrupt their function... University of British Columbia Department of Urological Sciences. Dr. Nabavi studies peritoneal mesothelioma...

...plasmids available at Addgene. We will still periodically focus on specific CRISPR plasmid tools more ...and an optogenetic tool that controls nuclear localization (LINuS). Blue light renders the NLS accessible...expression of specific Caulobacter genes using a catalytically dead Cas9 (dCas9) derived from Streptococcus...

...service. A small subset of AAV plasmids will have technical or legal restrictions that make them ineligible...be reviewed by Addgene for legal approvals and technical compatibility with our production process. This...found in our in-stock preps. If a request has technical considerations that may affect expected titers...

...hour long interview. I anxiously prepared for the call. The length of this interview was intimidating to...team and shows how they fit into the system. Specifically as a co-op, it is encouraged to explore different... a 9-5 As it turned out, Addgene uses something called “core hours”. Core hours consist of the time between...

...parts. Their efforts yielded a protein, which they call SpyLigase (Spy comes from the “S” in Streptococcus...able to grab on to proteins irreversibly in a genetically encoded way. SpyTag is nice and small, just 13...this tool? Howarth: Polyproteins are useful for mechanical study of proteins by atomic force microscopy ...

...the collection without getting overwhelmed by its scale: Look at the mEmerald variants first, followed by...fluorescently tagged versions of many proteins known to localize to specific cellular structures.The Davidson lab...you’re not sure where your protein of interest localizes in the cell, use these markers as your guide. ...

...American Cancer Society, or maybe you think about a local food pantry or community arts organization. Many...services we provide like quality control, curation, technical service and open-access educational resources....decisions at Addgene If you’re ever curious what specifically drives the decision making process at Addgene...

...sometimes no culprit can be found…. In our lab we call this mysterious source of failure the lab gnome....There must be some way to use LEGOs, which are practically universally loved (except for those rare times...children who have no preconceived obstacles, optimistically grasp at limitless potential, and who sometimes...

...wavelength of light. This response is immediate, on the scale of milliseconds, and can precisely link a neuron's...control over timing. Because chemogenetics relies on chemical activation and the diffusion of drugs throughout...require a permanent intracranial implant to be surgically installed to allow for light delivery. Chemogenetics...

...CRISPR array. The CRISPR array is composed of identical repeat sequences and variable spacer sequences...involve different proteins, but many of them are called Cas, short for “CRISPR-associated.” The most famous...integration into the array, these sequences are called protospacers. Repeats: Bacterial DNA that appears...

...let curiosity guide your intellectual journey. Practical approaches One approach to achieve this goal ...chemistry, even if your primary focus lies within a biological lab. Engaging in such seminars exposes you to...ourselves moments of release and mental freedom. Practical approaches Engaging in mindful meditation, taking...

...required to complete a six month work experience called a co-op to receive a degree. Northeastern compiles... occasional TikTok. I was also responsible for typical entry level tasks such as stuffing new depositor...connections with coworkers seem, in retrospect, nonsensical. I have found the people at Addgene have become...

...you covered! His His tags, often called polyhistidine tags, typically include six consecutive His residues...(Fox and Waugh, 2002). The tag can also be proteolytically cleaved after purification, so as not to perturb...

... While there were software tools available to biomedical researchers manipulating DNA sequences on a daily...different microbial strains. It proved to be impractical to catalog every variant of a feature. Instead...that tolerates occasional mismatches or indels. Empirical tests indicated that a reasonable rule is to require...