...awards to early career scientists using fluorescent proteins in their research. These awards help early career...biosensor fuses MPST to roGFP2. Because roGFP2 contains two cysteines near the chromophore that are susceptible... to better understand the processes that happen inside the cell,” says Pedre Pérez. Figure 2: ...changes following opioid withdrawal, providing insight into new methods for treating patients in recovery...indicator which had improved performance in live mouse brains. Currently, she is using the improved voltage indicator...

...destination vector + insert may spontaneously religate, this transient construct retains functional Type IIS...in the final construct. The destination vector contains sites with complementary overhangs that direct...complete with helpful tips and tricks, see The Sainsbury Lab website or Engler & Marillonet. Golden Gate...technology has adapted Golden Gate cloning for inserting the appropriate oligonucleotides specifying a ...

... they bind to a class of Argonaute proteins. The Argonaute proteins remove one strand of the siRNA and...cDNA), and then use PCR and molecular cloning to insert the cDNA into the desired plasmid backbone. The...the process is a little more complicated. These strains are more picky eaters and refuse to eat liver paste...Sánchez lab protocol also provides step-by-step instructions for the “the bravest and more curious” students...

...mutation in the evolved TadA domain. Because ABE7.10 contains a heterodimeric fusion of wild-type TadA and TadA7.10... the evolved TadA region. The ABE8.x-m version contains only the evolved TadA and is ~500 base-pair smaller...evaluate further.  The creation of accidental insertions and deletions (indel) by the catalytically impaired...more than 90% compared to ABE7.10. Choosing Cas9 proteins to increase the targeting scope of adenine base... Cas9 in the ABE complex with PAM-variant Cas9 proteins: the S. pyogenes NG-Cas9 (PAM: NG) to create NG-ABE8...

...challenge is finding relevant papers. The latest installment of the Early Career Researcher Toolbox will highlight...search that’s constantly updated. This Twitter list contains >300 Twitter literature bots, including a bot ...AI-backed literature search developed by the Allen Institute for Artificial Intelligence. Semantic Scholar ...Initiative. Meta is not a traditional search engine. Instead, you create your own research feed by entering ... papers are published, similar to Facebook or Instagram. Key features Setting up a personalized feed is...

...distinct proteins and result in unclear transcriptional consequences (Sprujit 2013). Enzymatic domains tethered...are a number of drawbacks to this approach: TET proteins are not demethylases; they are dioxygenases that..., Charlton et al. 2020). Most importantly, TET proteins affect transcription independent of their enzymatic...overlap the CpG of interest. Having the CpG directly inside the gRNA sequence is a great guarantee that DNMT1...

...secondary antibodies instead (B).   Since a polyclonal primary antibody contains a mix of different...antibodies used for detection bind to non-target immunoglobulins naturally present in the tissue sample. There...antibody fragments to block the endogenous immunoglobulins and reduce the background staining but they...Biological systems tend to be complex with a variety of proteins interacting. Experiments tend to be equally complex...

...first make your poster. While a poster generally contains the same sections as a primary research article...in your poster. This is a section where you can insert your references and contact information, and it...conclusions, and acknowledgements sections are inserted. The advantage of this style is that it will display... a poster vertically rather than horizontally. Instead of displaying your information as columns, you ... poster, written and graphical information are inserted into larger circles or ovals, that are arranged...

...use a standard strain, like DH5α, grown at 30 °C instead of 37 °C. If you’re unsure about the integrity ...prefer to store freshly harvested virus at 4 °C instead of storing it briefly at -80 °C. If troubleshooting...option may be to avoid freezing altogether, and instead to use the virus immediately upon harvesting. Interestingly...appear as a white pellet. The pellet can then be rinsed and resuspended in cold sterile PBS.  Alternatively...of ensuring viral production is to perform PCR against components of the viral vector using the harvested...

...Director of the National Human Genome Research Institute (NHGRI) of the NIH, started off with the first...will lead to advances in human health. It was inspiring to hear how the genome project has already had...enzymatic machinery (i.e. RNA replication with no proteins). The chemistry is complex and this work requires...desribed how P. furiosus has systems to defend against both DNA and RNA viruses - 2 cas systems regulate...system. His lab is also exploring the use of cas9 proteins from different species for genome engineering....

...—an optical insulator called a ferrule (below). The fiber optic cable/ferrule is inserted into the target...stimulation guarantees that the majority of the opsins will receive sufficient quantities of photons to...although you may be able to purchase one pre-made instead). Laser stimulation         In the video above...: closed/light receptive, open, closed/light insensitive. If you deliver a continuous stream of photons...

...practice: Rehearse your presentation with people inside and outside your field. Your colleagues, lab mates... tack up your poster seconds before the session begins. Plus, you’ll have extra time to practice at the... in to the next figure. Remember your outline: Instead of getting bogged down in the minutia or coming... practicing with your colleagues, ask them to brainstorm potential questions you may receive. People often...in to thirty-minute-long presentations, which constrains the number of posters you can see. If you are...

...variance of size of plasmids in your library (Are the inserts of different sizes?), and the desired coverage ...100x coverage of your library, and your library contains 5,000 plasmids, your goal for your transformation...replicate faster. For this reason, if your library has inserts of different sizes (such as a transcription factor...library is of a smaller size with a single size insert, liquid media can be faster and easier to work ...protocol paper by the Zhang lab, and the Broad Institute’s general pDNA Library Amplification protocol are...

...sensors. Given the expanding palette of fluorescent proteins and the availability of powerful imaging technologies...modules used in this method include fluorescent proteins, promoters, enhancers, polyadenylation signals...combined to create a single polycistronic 15 kb insert. The final construct was then introduced to HeLa...later combine all four constructs into one 15 kb insert. Table 1: MXS Constructs for Labeling Cellular ... fluorescent proteins...

...previous books focused primarily on fluorescent proteins and their applications, tackles much broader issues...from mildly surprising to genuinely shocking. Mainstream media outlets have addressed CRISPR frequently...science produced by the quieter, more reserved Franklins and Prashers will find it increasingly difficult...Addgenies Joanne Kamens, Marcy Patrick, Aliyah Weinstein, Fabienne Furt, and Quintin Marcelino for their... Eric J. Perkins...

...pastoris) because it secretes fewer of its own proteins, making the purification process simpler. Due ... the production of Fab fragments where variable chains are secreted into the periplasmic space to be oxidized...SDS-PAGE gel. Once stained, the heavy and light chains become visible alongside your ladder and a protein... density of the antibody bands can be measured against the protein standard and the concentration may ...Any bands on the gel beside the heavy and light chains can also be measured. Contaminating bands lower...

... for instance, a recent preprint released by the Boehm and Beroukhim labs at the Broad Institute of MIT...MIT and Harvard, Dana-Farber Cancer Institute, and Koch Institute, where an exploration into the effects...introduced an ancestry bias into their experiments instead of a true correlation.   It turned out there was...

... thanks to our guest blogger, Jessica Roginsky. Jessica Roginsky leads the scientific support team at ...This article was contributed by Jessica Roginsky, Scientific Support Lead at Synthego. Article source...genomes that have been successfully modified with insertions or deletions (indels) and then characterizes ...each bar of the indel plot shows the size of the insertion or deletion along with the percentage of genomes...

...collaborations. During my graduate studies at Karolinska Institutet I started working with other groups. First...minutes. Get to know your collaborators from other institutions personally on conference calls and make sure...learning and, in my experience, mistakes and insecurities are more easily excused. When you reach out ...Collaborate: An Imperative for Graduate Students" from Inside Higher Ed Additional Resources on the Addgene Blog...

... of all the proteins in the class that showed the relative sizes and the conserved domains. This information...highlighted a few dozen things that I didn’t know about. Instead of reading a paper and getting my bearings, I would...summarized how frequently each member of a class of proteins was mutated, as reported in various studies. We...sense of the similarities and differences among proteins in the class. Offer your perspective It doesn...

... This post was contributed by Ryan Watkins, Professor at George Washington University and developer of...in scientific journals flies under the radar and gains little notice. A 2009 research study found that ...influenced by research in other disciplines. But there remains a great deal to learn from the science of others...platforms (such as social networking, visual appeal, instant sharing across multiple other sites), I created...and let the world know about your science! Ryan Watkins is a professor at George Washington University ...

...DNA N-glycosylase (UNG) enzymes. Various other proteins included in different CGBE tools allow the researcher...a similar tool shortly thereafter, using mouse instead of human AAG (remember, MPG and AAG are the same...a more specific A → C base editor (ACBE), they inserted the deaminase into the C-terminal domain of Cas9...excises inosine can be modified to target guanosine instead, perhaps analogous engineering of UNG, which excises...its homologs) has more than one name. Indel Insertion/deletion A type of unwanted DNA edit. AYBE, ...

...cassette contains the major structural gene gvpA marked with EBFP2 and the second cassette contains the P2A-linked... be easily expressed heterologously by bacteria inside the body. So we screened gas vesicles gene clusters...bARGSer-expressing E. coli. The GV-specific ultrasound signal inside the tumors was overlaid with the anatomical B-mode...which seemed overexpressed compared to accessory proteins. Excess gvpA plasmid added into the transfection...

...Mashiko et al. 2013). The pCAG-EGxxFP target plasmid contains overlapping 5′ and 3′ EGFP fragments under the...MCS) between EGFP fragments. The pX330 plasmid contains humanized Cas9 expression cassette and sgRNA expression... surrounding your gRNA. Once amplified, you can insert this region into a pCAG-EGXXFP plasmid using standard...techniques (The XX indicates where your target is inserted, disrupting the EGFP fluorescent signal, for details...give specific details on what exactly has been inserted or deleted. Each lab will use a slightly different...

...Science (DBCLS),  National Cancer Institute, Public Domain, National Institutes of Health, Wikipedia user Aimecie...isoenzymes and genetic polymorphisms specific to their origins. This technique is still used today to test for...working cell bank has been made by a research institution (an excellent practice), or maybe cells have ...Brendan P., Walter A. Nelson-Rees, and Grover M. Hutchins. "Henrietta Lacks, HeLa cells, and cell culture...

... the study of neurological diseases, such as Parkinson’s disease (PD). PD is primarily characterized by...scientists to easily observe fluorescently labelled proteins and tissues to better assess developmental processes...of the pancreas and produce different levels of insulin (Hesselson et al., 2009). The worm (Caenorhabditis...From the baker to the bedside: yeast models of Parkinson's disease." Microbial cell 2.8 (2015): 262. PubMed...quick guide to Drosophila Download the fluorescent proteins 101 eBook Read our plasmids 101 blog series Resources...

...or projects is a common source of conflict. For instance, at Addgene, there are two teams whose primary... will not be great at resolving conflicts.  Institutional Knowledge: Of the tools I’m mentioning here,...accomplish, but there is a potential for long-term gains when you can do it. If Party X proposes solution...have used your empathy, listening skills, and institutional knowledge to resolve conflicts. Conflict in ... Eric J. Perkins...

...Addgene Advisory Board member, and Institute Scientist at the Broad Institute, John Doench. CRISPR technology...prime editing For a specific edit, such as the insertion of a fluorescent tag or the introduction of a ...the Genetic Perturbation Platform at the Broad Institute and has worked with many Addgenies to help improve...10.1038/s41576-018-0059-1 Veres A, Gosis BS, Ding Q, Collins R, Ragavendran A, Brand H, Erdin S, Cowan CA, Talkowski...

...Zhang, J., Chen, C., Papworth, G., Watkins, S., Trucco, M., Robbins, P. D., Li, J., & Xiao, X. (2006).... sometimes vary between target species or even strains. Pro tip! Getting a vector into a particular cell...growth factor receptor 1 (FGFR1)3 αVβ5 and α5β1 integrins3 Hepatocyte growth factor receptor (HGFR)3 Laminin...tropism varies both between species and even between strains of one species, so be sure to check the literature...

...the genome. When using piggyBac™, the cargo is inserted into the genome fully intact, while, with a standard...excision-only piggyBac™ transposase (PBx), which contains mutations making it competent for excision, but...specific point mutations by gene editing. First, instead of using piggyBac™, you could use recombinase technology...been suggested to cause cytotoxicity [5]. Second, instead of using a donor-plasmid, you could use a single-stranded...for the isolation and sequencing of retroviral insertion sites.,” Nat. Protoc., vol. 4, no. 5, pp. 789–...

...sciencedaily #instascience #ladies #research #cardiovascularresearch #parkinsonsdisease A post shared ...red nosed reindeer.       View this post on Instagram     Eppi the red nosed Reindeer! 🦌🦌 as you.... If we've tagged you it's because we love your Insta!! #LILCChristmas . . #lab #ladiesinlabcoats #Christmas...

...blog: Plasmids CRISPR Viral Vectors Fluorescent Proteins Science Careers You can use the dropdowns in ...awards that we offer there. Each topic category contains a overview page to help you get aquatinted. Here...comments below. We’ll track down the story, get the inside scoop, and keep you up-to-date. Be the first to...articles on the Addgene blog by subscribing to instant alerts below.  ...

...resources are not. Through its flagship program, Instrumental Access, Seeding Labs partners with donors to...opportunities, publishing assistance, and more to Instrumental Access participants. Like Seeding Labs, Addgene...plasmids to research labs that have received Instrumental Access equipment from Seeding Labs and that ...Global Plasmid Distribution Check out the List of Institutions that Deposit their Plasmids with Addgene ...

...between homologous pairs before viral packaging even begins. This spontaneous recombination persists even in... when the optimal time to conduct an experiment begins. This can save you time in the long run since not...accuracy and reproducibility of the data and can instill greater confidence when interpreting the outcome...https://doi.org/10.1128/jvi.02213-17 Fischer KB, Collins HK, Callaway EM (2019) Sources of off-target expression...recombinase-dependent AAV vectors and mitigation with cross-over insensitive ATG-out vectors. Proc Natl Acad Sci USA 116:27001...

...plasmids Anti-CRISPR proteins for optogenetic control of CRISPR-Cas9 Anti-CRISPR proteins that inhibit type...identified Cas14, a family of small DNA-targeting Cas proteins. Cas14 targets and cleaves ssDNA which they have...Now Dominik Niopek’s lab has adapted anti-CRISPR proteins for optogenetic control of CRISPR-Cas9. By combining...

...In the third installment in our podcast series, we chat with new Addgene Board Member, Michael Koeris... Koeris did his graduate work in Professor Jim Collins' lab (then at Boston University, now at MIT) where...Professor Timothy Lu (a graduate student in the Collins' lab at the time) got the idea to engineer bacteriophage...1:25 - 6:19 : Dr. Koeris' graduate work in the Collins' lab 6:19 - 8:26 : Dr. Koeris' reasons for wanting...

...Transformation grade DNA  Transformation is the process of inserting exogenous DNA in a microorganism. The amount of...cloning includes a set of techniques that are used to insert recombinant DNA from prokaryotes or eukaryotes ...from other nucleic acids (genomic DNA and RNA), proteins and chemical contaminants that have been used ... grade DNA. Transfection is the process of inserting an external plasmid in mammalian cells and it ...

... accomplished by using, for example, red light instead of blue to excite a molecule of GFP. Because red...a host of tools and opened doors to many great insights, it can be overwhelming to a novice microscopist...from a well-established microscopist at your institution when heading into a new set of experiments. ...Lightsheet Microscopy Choosing Your Fluorescent Proteins for Multi-Color Imaging Choose the Brightest Fluorescent... fluorescent proteins...

...wider community of users.  We also made scientific gains by adding long-read sequencing capabilities to complement...quality control. Through our partnership with the Institute for Protein Innovation (IPI), we expanded a growing...contributions to key research papers are already inspiring higher standards in antibody use (Ayoubi et al...R. A., Virk, H., Laflamme, C., Houston, D. W., Polinski, N. K., Meijers, R., Levey, A. I., Saper, C. B...

...Technique: Probe Phage Genomes for Host Binding Proteins & Running for Rare Disease, Running for FOP, Running...Technique: Probe Phage Genomes for Host Binding Proteins," was contributed by guest blogger Jessica Sacher...describes a technique you can use to find phage proteins that bind to your bacterium of interest. For those...an ingenious but simple technique to screen for proteins that could be useful for a wide variety of reasons...

... suffer any less acutely.  The Rare Genomics Institute works to help those who suffer from rare diseases...Through their BeHEARD Award, the Rare Genomics Institute partners with companies to provide technological...Elia Di Schiavi is a Research Scientist at the Institute of Biosciences and Bioresources in Naples, Italy...interactions with other genes. He will use fluorescent proteins to monitor the localization of these genes, CRISPR...

...capsid proteins that determine the AAV serotype. During transduction, the viral capsid proteins bind to...Education and Development workshop aimed at brainstorming strategies for using combinations of AAV-based... to the target cell’s surface proteins and this interaction determines the specificity and efficiency ...

...evaluate the impact of their work (H-index for instance), but these rely upon other researchers being ...plasmids that can be used for specific purposes. For instance, on our Optogenetics Collection page, you can ...containing plasmids for expressing different types of rhodopsins and sensors as well as references and links to... Blog Post on Our Citations Feature Browse More Inside Addgene Posts Additional Resources on Addgene....

...the presence of scientific heroines is always inspiring. I was especially excited to attend this event...genome. Their award acceptance addresses were inspiring! We also got to hear more from them during a panel...especially thanked Martin Jinek and Krzysztof Chylinski (who coincidentally speak the same Polish sub-...to generate mice with genes knocked out in the insulin signaling cascades in only 4 months compared to...

...does increase the chance of false-positives, as proteins may appear to be binding at particular genomic...Nuclei are then incubated with a primary antibody against the protein of interest. The Protein A fused MNase...G (IgG) thus targeting MNase to antibody bound proteins (Figure 1). Importantly, since Protein A has weaker...interactions Learn about other types of fusion proteins Study chromatin interactions with CAPTURE Additional...

...Microbiology (ASM) Scientific Writing and Publishing Institute. Writing is the cornerstone of any scientist’s...and the ASM Scientific Writing and Publishing Institute (SWPI) is an extremely beneficial guide to becoming...Di Rita, and, last, homework assignments. The instructors are leaders in the field of microbiology with...writing everyday Overall, the most important instruction the classes taught me was to write every day....

...recycled. This service does cost money, but your institute may still be interested as it offsets the cost...how to start a glove recycling program at your institute! What should I do in the lab to help the environment...back. Start a glove recycling program at your institute! Universities and companies have the potential...posts and updates from Labconscious. If you’re inspired by this post then I challenge you to take to action...

...and Leaders. I like her use of the term “cycles” instead of levels because it emphasizes the importance ...started and then step back to let others take the reins. They work alone much of the time, depending on ...it is the leader’s job to provide the driving inspiration, to create a productive culture and to correct... by Peter Fuda and Richard Badham Leadership Insights for Engineers, Scientists and Computer Professionals...

...molecular biology to isolate, multiply, or express the insert they carry in the target cell. These vectors allow...purify it – and you do not have a specific antibody against the protein available – you can always add an epitope...mammalian expression, a multiple cloning site to insert YGOI, a bacterial selection cassette for growth... collection of vectors carrying several fusion proteins. Pick your promoter, selection cassette and visualization...

...of the Ocular Genomics Institute, and an Associate Member at the Broad Institute of Harvard and MIT. ...In 11th grade she heard a talk about Z DNA that inspired her to major in molecular biology in college. ... of Oncology Target Discovery at the Novartis Institutes for Biomedical Research. Drug development was...