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...and inactivated transgene expression in neurons.  PubMed PMID: 2286602. PubMed Central PMCID: PMC3406316...sites directs the Cre recombinase to invert, translocate, or excise a DNA fragment. The availability of...creative ways. Today’s post will focus on one such strategy--the FLEx switch--which utilizes recombination...use two different pairs of target sites for this strategy to work effectively. Both loxP and lox511 are ...infected and would have the potential to express the transduced reporter gene. Generating a FLEx switch to control... orientation in the presence of Cre. A similar strategy can be employed for "Cre-off" (i.e. your gene ...O, Chambon P, and Ghyselinck NB. A directional strategy for monitoring Cre-mediated recombination at the...

...Interactions Difficult to detect weak and/or transient PPIs. Many PPI identification techniques rely ...often prevents the identification of weak and/or transient interacting proteins. No spatiotemporal control...BioID or Split-BioID, cells are lysed, protein extracted, and labeled protein interactors purified via ...BioID, is also useful for identifying weak and/or transient protein-protein interactions since the biotin ...speed of labeling. BioID requires 6-24 hours for tetracycline induced expression of the BirA* fusion protein...Split-BioID, Schopp et al allowed 24 hours for tetracycline induced expression and biotinylation of interacting... communications. PubMed PMID: 28585547. PubMed Central PMCID: 5467174. Find the Split-BioID Plasmids ...

...histone modifications, which can influence the transcription of genes by RNA polymerase II, or by the addition...methylcytosine in CpG islands makes  genes less transcriptionally active. The role of epigenetics in studying... before their adolescence passed on a heritable trait to their grandsons that decreased their likelihood... while still in their mother's womb passed on a trait to their granddaughters that reduced their lifespan...of tumor suppressor genes can repress their transcription and inactivate them. Global hypomethylation,...restriction enzyme digestion, DNA adenine methyltransferase identification, and bisulfite sequencing. ...Essentially this reporter construct consists of one substrate (the histone-derived peptide) which can localise...

... scientific training was put to use as we explored ideas, planned out potential strategies for pursuing...with a similar conundrum – she had the skills, training, and motivation, but there very few jobs available...our ideas got pretty good. We started doing extracurricular research on some of them to see how feasible...SynBio Axlr8r We limped along with very little traction for quite a while, and even had some serious self-confidence...and another $30,000 in mentorship and business training. The money is a drop in the bucket against the... the technology we will need, and the business training to make the most of it. It has also allowed us...below. We’re happy to answer questions about this transition (so far) and what we’ve learned.  Thank you ...

...expensive exogenously-added substrate, chromoproteins don’t require other substrates for screening. Thus chromoproteins...visible light to give colors in ambient light. In contrast, the widely used green fluorescent protein (GFP...bright green fluorescence when exposed to blue to ultraviolet light, whereas chromoproteins do not fluoresce...anemone’s red colored tentacles resemble worms which attract fish for them to prey upon (Figure 1). Figure... a gene encoding a blue chromoprotein, after transforming them into your cells, you can simply look for...imaging microscopy-based Förster resonance energy transfer (FLIM-FRET) in living cells (Figure 4). They created...by modifying the surface of the chromoprotein Ultramarine with hydrophilic amino acids to reduce non-specific...

...IN THIS POST HAS BEEN RETRACTED AND FOLLOW UP STUDIES HAVE FAILED TO DEMONSTRATE GENOME EDITING BY THIS...used by respondents (transfection method, amount of each component per transfection etc.). Take Pooran'...an ssDNA guide. Therefore, researchers must co-transfect cells with 5’-P-ssDNA guides and an NgAgo expression...delivery methods. Researchers can conveniently transfect/inject cells with in vitro preassembled Cas9/gRNA...researchers will have to carry out repeated transfections with the ssDNA guide, although many may find... an ssDNA guide. It is possible that, during translation, only a small fraction of nascent NgAgo protein...2014): 1262-1278. PubMed PMID: 24906146. PubMed Central PMCID: PMC4343198. Resources on the Addgene Blog...

...Melanie Fox, founder and executive director of Central Indiana Science Outreach and a Postdoc at Indiana...student culminated in my founding a nonprofit called Central Indiana Science Outreach, or CINSO. We organize...experiences in grad school, though often called “nontraditional,” helped prepare me to start CINSO. Here, I... from these contacts, I would have quit my “nontraditional” career path long ago. Use your analytical ...an excellent resource that has gained a lot of traction with researchers. It is an excellent way to meet... board. Although my goals may not have been “traditional,” I was able to fully take advantage of my graduate...currently the founder and executive director of Central Indiana Science Outreach and a Postdoc at Indiana...

...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...

... 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 ...

...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 ...

...Functional Screens New Tool for Lineage Tracing: The ClonTracer Library...that can be used for applications like lineage tracing or parsing the effects of expressing multiple genes...

...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...

...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 ...

...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...

...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...

... 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 ...

...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...

...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...