...a variety of imaging conditions (155 μs and <0.1 °C in optimal conditions). While B-gTEMP itself is a ...of interest and co-expressing a cytosolic ZtSH2 reporter, they observe fluorescence localizes to the plasma...And orthogonal sensors with different ITAM/SH2 reporter pairs allow multiplexed measurement of different...plasmids here!   Farahani PE, et al. pYtags enable spatiotemporal measurements of receptor tyrosine kinase signaling...10.1101/2022.08.13.503850.  iGECInano NIR calcium reporter Near-infrared (NIR) fluorescent proteins promise...photochemical characteristics than previous NIR calcium reporters iGECI and NIR-GECO2G, and its small size (~1500...sensors Whatever your analyte of interest, it’s important to choose a biosensor that has a binding affinity...

...in fewer cells being considered for the analysis. (C) A more liberal gate allows more cells to be considered...signals. For this, you’ll need to gate based on the proportion between H and A (or W and A) within the same ... just as fine (Figure 4). As H and A scale in proportion (while H and W do not; see Figure 2), single ...                                                                                                         B.    Figure 4: (A) Using the proportional relationship between FSC-H and FSC-A we can ...

... coli." Scientific reports 5 (2015): 15096. PubMed PMID: 28060411. Thomason, Lynn C., et al. "Recombineering...merely co-opted toward that aim (i.e. sacB). Importantly, to prevent activation of toxins before the second...

...3):937-948. doi: 10.1016/j.ymthe.2020.11.009  Guo C, Ma X, Gao F, Guo Y. Off-target effects in CRISPR/...better to reduce off-target effects, but it’s important to not let ‘perfection’ get in the way of ‘good... score their probability of off-target events: CRISPOR, Cas-OFFinder, CCTop, and many more. While such...

...composed of Venus fluorescent protein, the Troponin C Ca++ binding domain, and NanoLuc® Luciferase. CalFlux...has dubbed these fusions LumiFluor Reporters. The LumiFluor reporter constructs were designed to be packaged... The researchers took a FRET reporter and adapted it into a BRET reporter by using NanoLuc® Luciferase...share your constructs from lab to lab. Promega supports the Addgene mission statement: Accelerate research...(FLuc) and Renilla (RLuc) luciferase. This is important not so much for how bright you can make a reaction...couples NLuc to fluorescent proteins to make better reporters and the second couples NLuc to fluorescent proteins...host cells. The fluorescent moiety of the BRET reporter was used to sort successfully infected/expressing...

...10.1016/j.cell.2013.04.025 Zhang X, Liang P, Ding C, Zhang Z, Zhou J, Xie X, Huang R, Sun Y, Sun H, Zhang...enrich and select for cells that had successfully incorporated the desired mutation, and then derive mice from...handling embryos ex vivo, the components can be electroporated into the oviduct of pregnant females, a technique...gRNA, Cas9, and donor oligonucleotide can be electroporated into the mouse zygote (Qin et al., 2015).  ... it a single nucleotide change, insertion of a reporter gene, or replacement of the murine sequence with... microinjectionist, who has the strategically important job of delivering the payload! Last but not least...-Mediated Genome Editing in Mice by Zygote Electroporation of Nuclease. Genetics 200:423–430 . https:/...

...repository 49:45 0 1:18:45 - Reagent Development - Steven C. Almo, Institute for Protein Innovation  1:18:45 -...findings can be predictably applied to future work. Importantly, no one should expect that every study should...of reproducibility for preclinical studies, as reported by drug companies attempting to apply the findings...and use computer based analysis techniques, an important component of reproducibility is making sure that...

...4354 . https://doi.org/10.1073/pnas.76.9.4350 Yang P-C, Mahmood T (2012) Western blot: Technique, theory,...while less protein leads to a thinner band. It is important to note, however, that you must include a loading...

...measure this effect, known as covariance. This is the “C” that differentiates these tests from ANOVA/MANOVAs...most effective and accurate way possible it’s important to select the right test for the job (and, uh,...

... Y, Miki T, Fujioka R, Uehara R, Tomioka A, Obuse C, Kubo M, Hiwatashi Y, Goshima G (2012) An Inducible...fluorescent reporter, directly couple silencing of your gene target to silencing of the reporter. This is...application of long or short dsRNA. However, it is important to note that naked and unmodified dsRNA elicits... Together, this excellent work highlights the importance of using orthogonal methods to confidently conclude...fusing your gene target sequence to a fluorescent reporter sequence (discussed below). Examples of these ...genome, whereas shRNAs are exogenously derived. Importantly, instead of the assortment of siRNAs generated...contains a target sequence against the fluorescent reporter in an inverted repeat orientation. Therefore, ...

... my senior year isolating pheromones from various C. elegans mutants by myself, I would have slowly gone...orchestrate the formation of high-functioning-but-temporary teams that have a specific purpose and finite ...year, or a few months, or even a few weeks. The important thing to remember is that every team should have...when I do come across one. Also, remember, one important feature that all projects have in common - and... What does a good team need? Besides that all-important sense of purpose, there are many more things necessary... function is certainly necessary, but equally important is making sure all team members understand what...their responsibilities are. That’s a subtle but important distinction. If I invite someone to be on a project...

...neuronal firing through stimulating phospholipase C, which releases intracellular calcium stores. There...Mimura K, English JG, Liu J, Inoue K, Kumata K, Seki C, Ono M, Shimojo M, Zhang M-R, Tomita Y, Nakahara J...Link opens in a new window) Vardy E, Robinson JE, Li C, Olsen RHJ, DiBerto JF, et al. (2015). A New DREADD...Haberman A, Graham J, Block J, Zhou W, Chen Y, Zhang S-C (2021). Human Stem Cell-Derived Neurons Repair Circuits...control expression of a chemogenetic reporter ensures that the reporter remains silent until a cell or tissue-specific...ligands can have high potency, which is potentially important for dosing in animal studies, made them attractive...agonists for PSAM4. A given PSAM is coupled with an ion pore domain (IPD) to form ligand-gated ion channels (...

...920-931 (2005). PubMed PMID: 16299477. 3. Eggeling C, Willig KI, Sahl SJ, Hell SW. Lens-based fluorescence...Harvard University. No matter whether you are a sports photographer at the Super Bowl, a medical technologist...the first fluorescence microscopy technique to incorporate optical sectioning. Optical sectioning refers...their fast movements. Depending on the sample, a temporal resolution on the millisecond scale may be needed...plane of excitation light that is thinner than the portion of the sample that is in focus. This technique ...resolution due to the lack of a requirement for high temporal imaging speeds. Standard fluorescence widefield...sectioning, imaging over large distances and high temporal resolution. Traditionally, this type of sample...

...system and its dynamics. References 1. Peters, Nathan C., et al. "In vivo imaging reveals an essential role... methods allowed immunologists to develop a spatiotemporal view of an immune response, but the study of...transgenic mice expressing fluorescent protein reporter constructs in the immune lineage. By specifically...than the standard Ti:Sapphire laser. It is also important to finely tune the excitation wavelength as it...using 2-photon microscopy as this hasn't been well reported for many fluorescent proteins. Additional fluorescent...

...doi.org/10.1371/journal.pone.0197185. Chamness, James C., et al. “An Extensible Vector Toolkit and Parts Library...sub-cellular localization signals, plant markers, and reporters (Engler et al., 2014). This kit is a great starter...upon (its ‘basics’ MoClo plant kit buddy). It is important to understand your experimental needs before choosing...into a multi-TU targeting construct (Cas9, sgRNA, reporter, etc.). This allows for the multiplexing of sgRNAs...basic biology of and gene regulation in the opportunistic pathogen Acinetobacter baumannii (Brychcy et...used successfully for protein localization and transport studies in HEK cells, recombinant protein purification...Multi-Kingdom Golden Gate Cloning Platform.” Scientific Reports, vol. 9, no. 1, Springer Science and Business Media...

...stored at 4 °C for short-term storage. For long-term storage they should be stored at -20 °C in aliquots...conjugated to fluorophores should never be stored below 4 °C. Certain isotype subclasses, such as IgG3, aggregate... after thawing and therefore should be kept at 4 °C. Record and track lot numbers for all antibodies used...necessary in cases where extreme specificity is important. Their epitope specificity allows binding to a...antibodies only bind a single epitope, so it is important to choose a monoclonal antibody that will recognize...using a secondary against a tag, such as His, incorporated in the sdAb. sdAbs can have weak signals due...with conjugating reactions are possible. It is important to research your reaction and buffer before conjugating...

...with pLKO.1 C. Cloning shRNA oligos into pLKO.1 C.1 Recommended materials C.2 Annealing oligos C.3 Digesting...Virus may be stored at 4°C for a few days, but should be frozen at -20°C or -80°C for long-term storage...., R is a purine (A,G), and Y is a pyrimidine (C,U). G-C content should be 36-52%. Sense 3’ end should ...annealed oligo from step C.2 20 ng digested pLKO.1 TRC-cloning vector from step C.3 (If you were unable ...Store at 4°C. m. Add 5 mL of fresh media containing antibiotics to the cells and incubate at 37°C, 5% CO ...at 37°C, 5% CO 2 overnight. Day 2: b. The target cells should be approximately 80-90% confluent. c. Dilute...solution is stable at 4°C for up to one year. The powder form of polybrene is stable at 4°C for several years...

...before you start. Homogenizer Vortexer -20°C freezer -80°C freezer Reagents Solution D (for Protocol Option...instead of incubating at -20°C for 1 hour, you can try incubating at -80°C overnight. Centrifuge for 20...centrifuge the sample(s) for 15 minutes at 12,000 × g at 4°C to separate RNA from the rest of the tissue/cell lysate... lysate. Pro-Tip Having your samples spin at 4°C helps reduce RNA degradation. If you don’t have access...Isopropanol to the extracted aqueous layer. Incubate at -20°C for 1 hour. Lithium Chloride (Option B): LiCl selectively...extracted aqueous layer to 2.5 M. Incubate at -20°C for 1 hour. Pro-Tips If you anticipate your RNA yield...

...minutes at 94°C. Denature for 30 seconds at 94°C. Anneal primers for 30 seconds at 55°C (or 5°C below Tm)....step is commonly performed at 94–98°C. Denature 30 seconds at 94°C: Continued denaturation of double stranded...for 1 minute at 72°C: The Taq polymerase has an optimal temperature around 70-75°C so this step enables...minutes at 72°C. Repeat steps 8-10 for 25-30 cycles. Final Extension for 5 minutes at 72°C. Run 2 μL on...requires free nucleotides [dNTPs; adenine (A), cytosine (C), guanine (G), thymine (T)] in an equal molar ratio... Program Initial Denaturation for 2 minutes at 94°C: This initiation step heats the double stranded DNA...stranded DNA. Anneal primers for 30 seconds at 55°C: The forward and reverse primers are stable within...

... can open unexpectedly at -80°C. Freeze the glycerol stock tube at -80°C. The stock is now stable for ...sample at -80°C. Frozen tubes are hard to write on and samples stored for long periods at -80°C can lose labels...are important for long-term storage of plasmids. Although you can store your plasmid DNA at -20°C, many.... Bacteria on an LB agar plate can be stored at 4°C for a few weeks. However, if you want to store bacteria...glycerol stock of bacteria can be stored stably at -80°C for many years. Protocol Video Watch the protocol ...for years, as long as it is kept at -80°C. Subsequent freeze and thaw cycles reduce shelf life. To recover...completely and will improve the shelf life. It is very important that you shake the glycerol before freezing (5...