...Fluorescent tools for mammalian cells Nanobody purification CRISPRi and CRISPRa system for hPSCs The CRISPR...activity with a fluorescent protein--no affinity purification required! The team used other molecular techniques...compartments in mammalian cells By Angela Abitua Genetically encoded fluorescent proteins are often used to...collaborators have an ongoing effort to improve genetically encoded fluorescent markers for labeling specific...authors generated and described several improved genetically encoded fluorescent markers tagged with mTurquoise2...beads. Workflow of nanobody expression and purification. Iwaki et al., Protein Expr Purif, 2020. ...dCas9 and the gRNA. This expression can vary significantly depending on transgene design and delivery. ...

...Within that research group, you will also typically receive technical mentorship from a ‘bench mentor’. In ...receive after you’ve found your mentor, though. Typically, you will identify a ‘scientific mentor’: someone...scientific project. Your scientific mentor is typically a professor who operates a research group. Within...to do? Walk through the protocol mentally (or physically where relevant) and identify any gaps or questions...bring them to your mentor. Pro tip! Consider physically walking through the protocol, by going to each...something. It can be intimidating to ask for clarification at first, but in the end everyone wins. Your...experiments. Unlike in lab courses, where everything is magically made up by helpful TAs or assistants, and sitting...

... the bacteria were injected, limiting their applicability. They also did not scatter ultrasound nonlinearly...contrast in E. coli. We deleted an unannotated hypothetical protein, improving expression, and named this...promoter pBAD and a low-copy number origin of replication (p15A) worked best under a wide range of inducer...signal inside the tumors was overlaid with the anatomical B-mode images in greyscale.   ARG expression...of the mARG plamsids into HEK293T cells using a chemical transfection reagent (e.g. PEI-MAX). Simply cotransfect...-specific ultrasound signal is in hot and the anatomical B-mode images are in greyscale. (scalebar = 2mm...authors: Marjorie Buss is a PhD student studying chemical engineering. Mengtong "Tom" Duan is a PhD student...

... line from a reputable distributor such as the American Type Culture Collection and freeze as many early...contamination. For those labs that prefer a more economical mode of detection, Uphoff et. al. outline a do... freshly collected lentivirus for downstream applications. While this ensures that the highest titer virus...virus. To circumvent this problem, transductions typically employ the positively charged polycation, polybrene...establishes the best conditions for specific applications, stick with them! Changes in such variables ...Ward T.H., Margison G.P. “Mycoplasma infection significantly alters microarray gene expression profiles.”...lentiviral and retroviral vectors.” Human Gene Therapy Clinical Development 25.3 (2014). 175-85. Pubmed PMID: ...

...several ways. Below is a table listing some of critical differences (adapted from Zhou and Melton, 2008...this process. Moreover, it is much easier to genetically modify cells during the reprogramming process...This opens up a wide range of possibilities in clinical situations. In cases where the objective is to...Kushi) Mukherjee is the Editor of Trends in Pharmacological Sciences, a Cell Press reviews journal. She...joined Cell Press to pursue a career in science communication and publishing after completing her postdoctoral...from Massachusetts General Hospital and Harvard Medical School. Connect with her on LinkedIn @ https://...PMCID: PMC2854424. 23. Burridge, P.W., et al., Chemically defined generation of human cardiomyocytes. Nat...

...variety of species, cell types, and research applications. Labeling neurons with Brainbow The Lichtman...mOrange2, EGFP, and mKate2 (red) to solve the problematically low fluorescent intensity of previous versions... Brainbow 3.1 and 3.2 (described in the same publication) are unique because by default, they are not ...level of these XFP is not strong enough for all applications. To resolve this, similar systems have been ...developed a version of Tetbow that incorporates chemical tags instead of XFP to optimize it for tissue ...architecture of a fluorescent neuron is visible. Chemical tags are more resistant to tissue clearing methods...neuronal circuits with fluorescent proteins and chemical tags." eLife 7 (2018): e40350. PubMed PMID: 30454553...

...insights into the complicated physical organization of the brain and other biological systems. Interested...deletions are defined using 2 loxP variants (only identical loxP sites can mediate recombination.) The deletions...of farnesylated derivatives enables labeling of delicate axonal and dendritic processes not previously ...Between Brainbow Systems   Brainbow variants and applications beyond the mouse brain In addition to improving...temporal control without the need for germline modification, and enables Brainbow to be used in a variety...homeostasis results from neutral competition between symmetrically dividing Lgr5 stem cells. Snippert HJ, van der...

...content of this article. Biosensors (‘biological sensors’) are biological tools that monitor a process or detect...a reporter signal. Reporter signals may be electrochemical or light-based, with luminescent and fluorescent..., and you should always check the associated publication for the specifics of your chosen plasmid. Types...Experiments Experiment  Detection Method Notes Quantification of a process Plate-based assays (plate reader...components of individual cells Separation/identification of fluorescently-labeled components Fluorescence-activated... Jessica Welch...

...attended both online and on site courses with the American Society for Microbiology (ASM) Scientific Writing...of any scientist’s career. We use writing to communicate our findings and share them with the world. If...simply writing down your data doesn’t guarantee a publication. There is an art to composing manuscripts and...field of microbiology with multiple high-profile publications. My classmates consisted of graduate students...sessions and reinforced our newfound knowledge with practical understanding of how to apply it. The personal...well as being critiqued added an extra level of application not present in the online class.   Four ways ...paper will be generated. After you complete a publication quality figure, produce the figure, its legend...

... some of the drawbacks. Specifically, viruses are able to infect and replicate in host cells, but they...gene therapy using Sleeping Beauty to genetically modify clinical-grade T cells to target CD19. Immunol... though not all, of these defenses. For some applications of genome engineering - such as certain forms...to integrate. Additionally, the TA sequence is duplicated near the gene of interest insertion site.   The...Anderson Cancer Center presented results from a clinical trial using the Sleeping Beauty transposase as...

...the less acknowledged perks of scientific and technical training is that these educational paths prepare...Foundation study showed that 57% of PhDs in US Biomedical workforce will NOT go into “traditional” academic...tech transfer, management consulting, science communication, policy and the diverse options in nonprofit...science is much like industry but the “product” is publications—the pressure to produce is the same You get ...and teach students Collaborations may be less “complicated” - for example, easy to initiate a collaboration...is different about non-academia? Scientific publications are a nice to have, not a need to have Must ...every 3-6 years—a short and exciting ride Complicated matrix structures are common Small integrated...

...protein is (localization) and how much there is (quantification). But what if you’ve got several interesting...inactivate the fluorophores in the sample, either by chemical treatment or photobleaching or by stripping out...primary antibodies that have been validated in IF applications (find IHC validated or ICC validated antibodies...yourself stuck with red-green images generated automatically from the microscope software, you can always...it, the secondary antibody may be binding non-specifically – try optimizing your blocking and washing conditions...informative image-based figures for scientific publications. PLOS Biology, 19(3), e3001161. https://doi....Germain, R. N. (2020). IBEX: A versatile multiplex optical imaging approach for deep phenotyping and spatial...

...Musicophilia includes case studies of relevant neurological atypicalities, including a patient with amusia - the...capture the attention of even the most skeptical reader of historical fiction (I can personally attest to...sometimes we don't."     - Jennifer Tsang, Science Communications and Marketing Coordinator Non-science favorites...Strange & Mr. Norrell by Susanna Clarke "A quasi-historical/fantasy fiction. Two dueling magicians in the...times."     - Aliyah Weinstein, Marketing and Communications Manager Addgene's book club Indeed, reading...

... to a method of physically joining multiple fragments of DNA to create a synthetically designed DNA sequence...-guide RNAs – but it also allows you to skip purification steps and end-repair steps with well-designed...fragment sequences and the polymerase used in the amplification. This tool allows instant updates as fragments...transforma­­­tion or as a PCR template. Some of the applications of the NEBuilder HiFi DNA Assembly Mix include...one single-stranded oligo, instead of the more typical two oligos, is more cost effective and eliminates...the limitations associated with otherwise more complicated assembly approaches, and opens the door for new...

... fused base editors (nCas9-BE), as Cas9 is mechanistically more favored for efficient base editing. They...been used for multiplex Cas9-nuclease editing. Typically, a single array encoding the tandemly assembled...released by endogenous tRNA processing machineries, specifically, RNaseZ and RNaseP. Although the Gao Lab demonstrated...packaged into viral vectors for potential in vivo applications or multiplex CRISPR screening.   Using tRNAs...multiplex genome engineering of crops or allow for sophisticated CRISPR screenings that would perturb the unprecedented...and efficient in plasmid and viral vectors. In applications when repetitive tRNA sequences must be avoided...construct DAP array for multiplex gene editing applications, we provide detailed manual and online design...

... necessary for many of these applications have lagged. Fixation, chemical staining, and light exposure...antibodies may just not perform as well in one application as another, so it is always best to have options...for mEGFP-HRas (from Plasmid 18662). See the Applications section on the Anti-GFP [N86/8R] page for more...30667360; PMCID: PMC6377228. Hyperfolder YFP- a chemically stable tool for microscopy by: Susanna Stroik...well-suited for the demands of advanced microscopy applications such as super resolution, electron, and expansion...Find YFP plasmids here!   Campbell, B.C., et al. Chemically stable fluorescent proteins for advanced microscopy...

...generation when bound by the dL5** FAP.  The iodine-modifications also shift MG-2I’s excitation wavelength to ...FAP-TAP system works. Find the Plasmids from the Publication Here Light-induced cell ablation with MG-2I-dL5...the main advantages of MG-2I-dL5** over other genetically encoded photosensitizers is that it works with... ROS because NIR light doesn’t excite natural biological chromophores found in cells.  He et al also show...compared MG-2I-dL5** head-to-head with KillerRed, a genetically encoded red fluorescent photosensitizer protein...Watkins, S. C., . . . Bruchez, M. P. (2016). A genetically targetable near-infrared photosensitizer. Nature...Chepurnykh, T. V., . . . Lukyanov, K. A. (2005). A genetically encoded photosensitizer. Nature Biotechnology...

...around these issues, but this solution wasn’t practical due to the low intensity of luminescent proteins...resulting in a fluorescent signal. Since their first publication in 2012, the Nagai laboratory has assembled a...multicolored nano-lanterns for use in various applications, including optogenetics, biosensors, and fusion...sensitivity and faster imaging compared to previous publications. They also developed calcium, cAMP, and ATP ...furimazine. In addition to the previously described applications, the enhanced signal of eNL allows their use...characterizing Nano-lanterns and eNL for many different applications. For long-term imaging, especially where photobleaching...not alter cellular physiology in your system. Optical sectioning cannot be conducted with luminescence...

...you're trapped in a pattern of small errors that significantly impacts your experiments. What do you do when...variables - your approach, your workflow, or your physical setup - to prevent the mistake, just like you ...differently.  Tools to Try  Checklists Found in surgical suites, air fields, and military bases, checklists...light-sensitive reagents” or “re-sterilize now.” Have a physical copy of the protocol when you work and make a ...time to do them carefully and thoroughly. Have a physical copy on hand as you run your experiments. Try ...your plate maps onto your plates, alongside your physical copy. If you’ve got messy handwriting, reach for...accuracy.  Bench setup Small adjustments in the physical organization of your workspace and your workflow...

...made of chemicals, which have an unfortunate tendency to react with other chemicals and biological structures...antibody out on the bench overnight. Do not panic! Typically, antibodies are okay if they’re left out overnight...then I would suggest either testing the antibody efficacy through a standard curve, another quick experiment... fluorophores, which work not by reacting to a chemical, but by emitting a specific wavelength of light...compensation.    Working with antibodies can get a bit complicated, but hopefully this helps you successfully select...