...wavelengths (See this handy emission spectra chart to find your antibody's spectra). Your cytometer will therefore...probes on the antibodies may have similar emission spectra, meaning they will emit fluorescent light at similar...be compensated for one another. This overlap in spectra is often called "spillover." For example, if your...BV510 and AmCyan have almost identical emission spectra but may be called BV510 on one cytometer and AmCyan...against APC-Cy7, the populations are in more of a straight line and are only positive for APC (Figure 1B)... plot in 2A, the populations are not perfectly straight, but the center of each population is in a line... line in the plot. However, because we can see straight lines in the populations in the other corners ...

... muscle contractions, and the control of voltage gated ion channels all depend on transmitting and sensing...insufficient level. The heartbeat – a muscle contraction – is directly related to the electrical impulse...propagated through multiple cells to do things like translate perceived signals to physical actions. Understanding...rhodopsin proteins were initially evolved for ion transport, not fluorescence, they are not nearly as bright...conditions, which are necessary conditions for penetrating deeper tissues depths (1 mm), important for certain...and emit signal from. This specific example is attractive due to the large number of sheer molecules and... this method have been to tag, cage, uncage, and trap the chemical fluor with the genetically encoded ...

...to find the ones you’d like to revisit. Strategically traverse the sea of posters If you come prepared...interest causes cancer, as well as therapeutic strategies to target it, than you typically would at a microbiology...keep you energized throughout the session, bring extra clips to hold up your poster, and pack a few business...seconds before the session begins. Plus, you’ll have extra time to practice at the actual venue. Introduce ...For most larger conferences, poster titles and abstracts are available online or in an app prior to the... through the list at the session so you can strategically use your time. Don’t have access to a list of...in to thirty-minute-long presentations, which constrains the number of posters you can see. If you are...

...control strain. A good negative control strain is an untransformed culture of the same strain of bacteria...screening colonies for the presence of an insert. Traditionally this has been done with restriction enzyme digest... colony PCR is designing primers. There are 3 strategies for primer design: 1) insert-specific primers...mutations.  There are lots of different cloning strategies, but regardless of which is your favorite, colony...control. A good positive control is bacteria transformed with the same backbone plasmid. If this control...Cloning Technique Learn to Perform a Bacterial Transformation Deposit Your Positive Clones with Us ...

...but they can also be expressed in bacteria and extracted with common protein purification methods. Read...below for a comparison of nanobody, HCab, and traditional IgG antibody structures. Functional comparison...microscopy. Their size also limits their ability to penetrate tissue. Because antibodies are large and bivalent...recombinant; Require animal sacrifice Poor tissue penetration Less resolution due to larger label displacement... do not require animal sacrifice Good tissue penetration Can be labeled with multiple dyes Greater resolution...Their small size also allows for better tissue penetration and decreases the distance between a fluorescent...Lee, C., Pleiner, T., Schliep, J.E., Stark, H., Trakhanov, S., & Urlaub, H. (2015). Nanobodies: site-specific...

...C-to-U and A-to-I transcriptome-wide off target effects observed in whole-transcriptome RNA-sequencing, ...RNA editing has multiple advantages over more traditional DNA editing systems; first, RNA editing doesn't...would convert adenine to inosine, which the translational machinery treats like guanine. This RNA editor...amount of off-target editing. In one direct transcriptome-wide comparison, REPAIRv2 reduced off-target... on the ADAR2DD residues that contact the RNA substrate resulted in 15% C-to-U editing activity. They ...the RESCUE system’s ability to edit endogenous transcripts finding up to 42% editing efficiency. They note... (2016):aaf5573. PubMed PMID: 27256883. PubMed Central PMCID: PMC5127784. Find plasmids from this paper...

...better access to intracellular targets. If your target is already on the extracellular side of the membrane...high a concentration.      Figure 2: Examples of different permeabilization strategies and when...after fixing your sample with a cross-linker. This strategy gives you the benefits of both reagents - cross-linkers...many membrane-associated proteins in place. In contrast, Triton X-100 and Tween-20 are examples of non-ionic...is suitable for antibodies that bind to the extracellular portion of a protein. B) Permeabilizing with...antibodies into the cell where they could bind to intracellular epitopes. C) Permeabilizing with a non-ionic... then methanol treatment could be an effective strategy. (This article on fixation has some more examples...

... ants become “zombies” and leave their foraging trail. They climb upwards until they reach a point where... the snail, the larvae reach the eyestalk and transform them into a pulsating display imitating a moving...larvae. The adult worms develop in the digestive tract and release eggs in the bird feces, which get eaten...grasshoppers and crickets. When it’s ready to transform into an aquatic adult, it forces its insect host...produces proteins which affect the grasshopper’s central nervous system: infected grasshoppers expressed...uninfected grasshoppers, including those linked to neurotransmitter activity and geotactic behavior (Biron et ...2005): 2117-2126. PubMed PMID: 16191624. PubMed Central PMCID: PMC1559948. Brusca, Richard C., and Matthew...

...best gRNA Figure 1: Validation of double strand break (DSB) mediated homology dependent repair ...repair (HR, homologous recombination; SSA, single strand annealing) took place and reconstituted the EGFP...site. (c) The pCAG-EGxxFP target plasmid was co-transfected with pX330 plasmids containing sgRNA sequences...efficacy of your gRNAs is then a simple matter of transfecting HEK293T cells with your modified pCAG-EGXXFP...fluorescence, the better that gRNA is at causing a double stranded break at the target site, allowing the two halves...plasmid DNA is much easier and doesn’t require the extra precautions needed when dealing with RNA (e.g. face...(2014): 132-141. PubMed PMID: 24253446. PubMed Central PMCID: PMC3875854. 2. Mashiko, Daisuke, et al. ...

...have since been retracted due to cell line contamination: Epithelial Mesenchymal Transition Is Required for...records allows you to track a cell line’s age and monitor any changes. Your tracking system could be as ...biochemical assays followed by in vivo testing. The mantra being “you can’t trust those whacky cell lines”...colleague. Time and money are wasted, papers are retracted and there is a loss of reputation that can impede... good recordkeeping, you need to have a way of tracking how many passages the cells have undergone and...variety of pharmaceutical companies including GSK, AstraZeneca-MedImmune, and Infors & Applikon. Due to his ...his high level of experience he now travels around Europe giving talks and seminars on cell culture and ...

...sample to that of a standard curve to extrapolate the concentration of antigen in the sample. A variety ...chemiluminescent substrates are available. Labs with limited equipment often opt for the chromogenic substrates since...For enzymatic signal detection, users provide a substrate that the enzyme converts into a detectable product...antibody. The reporter reacts with a provided substrate and produces a signal that is directly proportional... present in the sample and is calculated by extrapolating from the standard curve. Because the primary...enzymatic reaction between the detection antibody and substrate is proportional to the amount of antigen present...present in the sample and is calculated by extrapolating from the standard curve. Scientists often opt for...

...you will interpret and use the information. In contrast, a list of experiments or a laundry list of molecules...for or against a model is a standard scientific strategy. However, a robust research proposal will distinguish...mystery that motivates your research? This is the central idea. It should be at a high level, broadly understandable...is a significant need for healthy livers for transplants. Growth of new livers in culture could resolve..., neither of these can substitute for a liver transplant. My lab will combine bioengineered scaffolds ...delivery. Design simple figures and diagrams to illustrate your vision. Practice drawing and writing everything...create a sculpture or print a 3D model to better illustrate your point.  Practice the full chalk talk with...

...?  Plasmids 101: Transformation, Transduction, Bacterial Conjugation, and Tranfection...genes necessary for transfer initiation between bacterial hosts. Also, self-transmissible plasmids may have...determined by the different hosts that a plasmid can transfer to, replicate in, and persist in. Some plasmids...easily cloned in different bacteria, and can be transferred to various experimental hosts (Jain and Srivastava...scientific community, as they allow for simplified transformation processes, and can be used by scientists across...past this checkpoint.  The plasmid is self-transmissible, meaning that the plasmid is conjugative and...

...has been removed/mutated to neutralize the nuclease activity. For strains that still carry this gene, ...from the DNA. These apurinic sites if in double stranded DNA are repaired by base excision repair, but ...but can lead to mutations in single stranded DNA undergoing replication.   DNase on the other hand can digest...main causes of degradation during plasmid DNA extraction and purification from E. coli is the presence...the gene endA. Nowadays, the most common E. coli strains used for plasmid amplification are endA negative..., there are usually extra steps like heat inactivation, protein degradation, and/or the use of chelating...dilute your sample in water to reduce the EDTA concentration. Alternatively, if the priority is to directly...

...cells, and someone new to plasmids may need some extra guidance to understand the specific components that...survival, such as an antibiotic resistance gene. In contrast, plasmids utilized in the lab are usually artificial...plasmids to self-replicate within a cell make them attractive tools for the life scientist or bioengineer. ...for further study. Promoter Region Drives transcription of the target gene. Vital component for expression...cell types. Primer Binding Site A short single-stranded DNA sequence used as an initiation point for PCR...vector containing the newly inserted gene is transformed into bacterial cells and selectively grown on...Produce proteins that glow so that scientists can track their location or quantity inside a cell Plasmids...

...fun is building within the constraints given. And there were many constraints during the COVID-19 pandemic...looking, I found an inexpensive HDMI transmitter that could transmit a signal between a wall. There was ...impressive, but due to costs and availability, a traditional solution was completely out of reach for Addgene... things first was getting that wireless HDMI transmitter/receiver, to beam the camera’s image to my remote...then set up a monitor connected to the wireless transmitter and an iPad in the office next door (connected...with wireless video receiver, camera control, and extra hand sanitizer.    Cycle 4: Venturing into the...was a bit cumbersome, but it was mobile enough to travel around the office with me, and to move it out of...

...fluorescent protein that was derived from the tetrameric yellow fluorescent protein (LanYFP) from the ...fusion tag for a wide range of applications from traditional imaging to FRET and stochastic single-molecule...fluorescent protein mTPF1 is a monomeric version of the tetrameric cyan fluorescent protein from the coral Clavularia...Alberta and University of Oregon created mTFP1 by transforming E. coli with a gene library of approximately...approximately 484 variants, including some potential tetramer-breaking mutations. The library was then screened through...(2006): 531-540. PubMed PMID: 16859491. PubMed Central PMCID: PMC1698604. Ai, Hui-wang, et al. "Hue-shifted... 6.1 (2008): 13. PubMed PMID: 18325109. PubMed Central PMCID: PMC2292683. Shaner, Nathan C., et al. "A...

...sensor proteins, particularly for the intracellular imaging of transition metal ions, such as zinc, copper,...field is a series of fluorescence resonance energy transfer (FRET)-based calcium biosensors. Perhaps most ...firing in neurons and high resolution functional tracking of T lymphocytes, too, making them a versatile...developed and published in 2010 for visualizing the transcription factor cAMP-responsive element-binding protein...really important and when? That is not known.” Transition Metals Maarten Merkx of Technische Universiteit...the sensors involves plenty of trial and error. Transition metals also present challenges in that they are... are found in cells at much lower concentrations than calcium, requiring greater sensitivity. He and his...

...FPbase Fluorescence Spectra Viewer can be used to compare excitation and emission spectra when available, ...soon feature filter spectra and collection efficiency calculations.  With ~80 FP spectra (and rapidly growing...than is currently represented at fpvis (complete spectral information, accession numbers, sequences and ...thinking about FPs, it would be great to have a centralized, community-editable resource for storage and ...the largest available online collections of FP spectra. Every protein has a page with all the information...biosensors, protein evolution lineages, 2-photon spectra, more elaborate spectrum analysis tools, and a ...

... of the sensor. This “contrast” variant provides improved detection for tracking large populations of ...ppAAV-CKIIa-stGtACR1-FusionRed New tetracycline transactivators Plasmid Serotype Name 99120  AAV1...particular study, which includes comparing the transduction patterns of different AAV serotypes. To help... Biosensor AAV The ability to detect the neurotransmitters and other bioactive molecules is integral ...jGCaMP7c variant in AAV1. This variant exhibits high contrast between its resting state and its calcium-bound...fusion fluorescent proteins can affect DREADD trafficking and localization. The Sternson lab recently deposited...