...As evidenced by all the CRISPR publications, press, and plasmids out there, it’s obvious that CRISPR ...nickase variant, lowering Cas9 expression, and truncating gRNA sequences used for targeting; however, these...on-target and off-target activity at multiple genomic locations in HEK cells. Further testing of two of the mutants...PubMed PMID: 26628643. Find plasmids from this publication at Addgene. 2. Nishimasu, Hiroshi, et al. "Crystal...PubMed PMID: 26735016. Find plasmids from this publication at Addgene. 5. Chen, JS, et al. "Enhanced proofreading...PubMed PMID: 28931002. Find plasmids from this publication at Addgene. Resources on the Addgene Blog Read...

...repurpose popular GFP reporter lines for more sophisticated experimental manipulations, saving the time ..., 98% of TdTomato expressing cells were GFP+, indicating a robust, but specific system. T-DDOGs also successfully...systems are easily adaptable to neuroscience applications, including optogenetics, and they should make...Could Cas9-DOG be developed soon to allow genome modification only in a small subset of cells? Other proteins... PMCID: PMC4096992. Find plasmids from this publication at Addgene. 2. Tang, Jonathan C.Y., et al. “...PubMed PMID: 26258682. Find plasmids from this publication at Addgene. 3. Kirchhofer, A., et al. “Modulation...

...These properties allow for efficient signal amplification, making biotin conjugated antibodies good options...especially if you start to consider clinical applications). But there are a few other classes that we ...proximity ligation, immuno-PCR, and single-cell applications (Hegazy, et al., 2020; Niemeyer, et al., 2007...also conjugate antibodies directly to beads for purification purposes.  Coupling up - antibody conjugation...added to antibodies post-translationally. 4) Modifications made to antibodies, such as inclusion of non-canonical...Detecting Protein-Protein Interactions and Protein Modifications in Cells and Tissues in Situ. Curr Protoc Cell...

...detected with tyramide signal amplification. Tyramide signal amplification occurs when HRP converts a fluorescently...reporters (i.e. fluorescent dyes, HRP, etc. ) after purification. Antibodies are difficult for an individual ...culture media. Their His tags allow for further purification, if desired, but it's not required for their...some reporters are better suited for particular applications. Read on to learn more about each reporter or...Pros Cons Antigens HRP (P) Tyramide signal amplification of HRP Staining cultured cells and tissue sections...

...rounds of replication are initiated before the cell has divided. This means that genes located near the...problems. Rapidly dividing cells initiate genome replication more than once per cell division and thus, genes...genes closer to the origin of replication will be overrepresented compared to those further away. As a...maximal gene expression near genomic origins of replication, the stabilized promoters created by Segall-Shapiro...the control of IPTG. Because RepA is a plasmid replication initiator protein, they could alter copy number...

...choose well. What are their qualifications? Do they have certifications? How can they demonstrate their...  Check out Joanne's Reddit AMA I am a strong advocate for peer mentoring, but volunteer mentors are ...help you. A good coach usually will be a clear communicator and make direct suggestions for improvement ...coaching is nice if you can get it.  Because I am located in Cambridge, MA (and because we have a lot of ...she is especially excellent in coaching for communication skills. Industry transition is also an area ...

...members of their pathways, but where the kinases are located, their signal strength, and how long their signals...and MEK1 is active or “on.” Dronpa’s color thus indicates MEK1’s current state (Green when off, dark when...domains were inserted at sites homologous to the locations they were attached to in MEK. The photoswitchable...kinases good for? There are a number of potential applications for photoswitchable kinases.  Below are a few...signaling by using psMEK1 and an ERK kinase translocation reporter with a mRuby2 fluorescent reporter ...endogenous ERK, and its localization serves as an indicator of MEK1 activity. When MEK1 is active, it phosphorylates...

...days prior to the implementation date, our data indicated that, while 69% of all postdocs were expected ...sent, with the lack of transparency and poor communication of their plans (many postdocs - and in some ...to reach out to institutions with a statement indicating that we encourage them to keep raising salaries... little correlation with cost of living at the location of the institution. Continuing efforts to gather...Director of Future of Research. He is interested in advocating for and with junior scientists, to help them ... Bring Increased Postdoc Wages Read about the Advocating for Science Symposium and Workshop Learn about...

...scientific poster apart? First, a good poster should catch the audience’s eye and draw people in. That can ... sections should be as concise as possible, communicating only the most important results to your audience...have the advantage of being able to verbally communicate your work, so you don’t need every little detail...may choose to display it as rows. It can be more catchy because it is generally less common and will stand...advantage of being different and potentially more catchy, but it can be trickier to make figures fit nicely...relevant to the work you are presenting. The latter is catchy, but beware. A distracting background or clashing...

...chemical replication of RNA in the absence of evolved enzymatic machinery (i.e. RNA replication with no...Guangzhou and the Science City The conference was located in the modern, relatively new “Science City” area...and technology companies and start-ups choose to locate there. I made time to go into the city of Guangzhou...inheritance that might be less error prone than DNA replication. He too mentioned how “surprising” small RNAs...Steitz quipped that RNA in the ribosome is in a complicated conformation “not like DNA which has a boring...

..., its dimeric nature can interfere with some applications. Several labs have been working to produce monomeric... for molecular fusion and membrane-targeting applications. Nature Methods, 1–9. https://doi.org/10.1038...library generation and screening that led to identification and characterization of AAV.CAP-Mac. Image ...rodents and broad tropism in primates. Nature Communications, 14(1), 3345. https://doi.org/10.1038/s41467...proven itself a good option for essentially all applications. Addgene’s plasmid collection includes over ...clone (M2) has been widely used in a variety of applications. Find anti-DYKDDDDK recombinant antibodies here...

...experiences to undergraduate students (1, 2), but educators face significant challenges in designing suitable... become very different due to a whole genome duplication and extensive rearrangements in the S. cerevisiae...overexpression vector. This plasmid contains origins of replication and selectable markers that allow students to...met deficiencies (manuscript in preparation), indicating that Met protein functions have been largely ... research into undergraduate courses and in communicating science to the public. You can find out more...). Vision and Change in Undergraduate Biology Education: A Call to Action, Washington, DC. 2. President's...

...the best, or even which one will work for your application? Like many scientists, I have spent hours sifting...produced from a new set of animals), and wrong application (different experiments/conditions can change ...differently over time), compatible with the desired applications (such as immunohistochemistry (IHC) or western...colleagues! Dr. Wu and his lab members (like me!) are dedicated to creating a public and accessible database for...expectation and comparison on the antibody signal’s location. If a distinct imaged pattern does NOT align with...literature and is unrelated to the target protein, we categorize the antibody as having non-specific binding in...

... biological reagents have a ton of data on the catalog page. It’s not always super easy to navigate the...originally described in a peer-reviewed scientific publication. We love science, but we don’t have the resources...questions about an Abcam antibody. Have your catalog number ready. I've attempted to answer several ...Addgene support, make sure to mention the plasmid catalog number, or at least the plasmid name in your initial...be fairly fast, getting the full answer to a complicated question may take some time. If the customer ...message Answering customer service questions is a delicate balance of knowledge, patience, and diplomacy,...

...targeting anywhere in an exon. But things get more complicated if you’d like to use homology directed repair...They created a selection phage (SP) to carry catalytically dead SpCas9 (dCas9) fused to the bacterial polymerase...test xCas9 3.0-3.13, Hu et al. first restored catalytic residues D10 and H840. To ensure that the clones...loci displayed a broad range of PAM sequences, indicating they may be less predictable than SpCas9 off-... that we have only scratched the surface of modifications that can be made to Cas9. It is a pleasant surprise...editing rates. This PAM flexibility should expand applications that require very specific targeting, like homology...

...retroviruses ” (Cullen, 1992) because of their more complicated genomes. Here, we will mostly use the gamma-retroviruses...wild-type viral particle — and of the normal, replicative life cycle — can help you understand these seemingly...of a non-living viral particle, and how we as dedicated researchers can exploit it. This blog was written...Hutcheson for her assistance with lentiviral replication.  Resources and References More resources on...101: Types of Viruses Viral Vectors 101: Viral Applications Resources on addgene.org Addgene's Lentiviral...acid of the Bryan strain of Rous sarcoma virus: purification of the virus and isolation of the nucleic acid...

...RESCUE in therapeutic applications, the authors tested various dRanCas13b truncations that would allow packaging...longer stretches of duplexed RNA. Using an ADAR2DD catalytic mutant, they showed that editing is mediated by...into adeno-associated viral vectors, they tested truncated ADARDDs to see if they could shrink the construct... to deaminate cytidine and used dRanCas13b (catalytically inactive Cas13) to target the cytosine deaminase...construct for viral delivery. They found C-terminal truncations allowed the same or improved editing ability ...

...plasmid using standard cloning techniques (The XX indicates where your target is inserted, disrupting the ...effective ones to increase your chances of gene modification in vivo. Find Plasmids for Testing Your gRNAs...et al., 2014). The plasmid is sturdier than the delicate RNA and therefore there has less chance of contamination... the validation assay should give an accurate indication of how many pups will contain a mutation and ... months from the day you decide to do a gene modification experiment in mice to the day you have your ...reproductive biology and genetics. She loves to communicate the latest and greatest in scientific content...

...a protein’s stability or post-translational modifications, or study how a target protein interacts with...Protein A and Protein G are not suitable for all applications, such as protein isolation from serum. In this...capture antibody interaction and/or the downstream application. Typically, if the target protein will be detected...and an isotype control antibody. Alternative applications Oftentimes, an IP-appropriate antibody simply...identify DNA binding proteins involved in histone modification. Similarly, RNA IP (RIP) can be used to isolate...resources References:  Björck L, Kronvall G (1984) Purification and some properties of streptococcal protein...

...EthoVision. Consult the literature to find all the applications of optogenetics relevant to your field. Ultimately...Inhibition of neuronal activation is a little more complicated since basal neuronal activity in your brain region...technology is improving and may one day reach the sophistication of mouse transgenics. Everything that has been... and memory in opioid addiction and science communication/science writing. Derek is also a pianist and...rat lines: tools, techniques, and optogenetic application to dopamine-mediated reinforcement." Neuron 72.5...sensitivity optical neural silencer: development and application to optogenetic control of non-human primate cortex...