.... Many thanks to our Guest Blogger Kristian Laursen! Kristian Laursen is a researcher at Cornell University...This post was contributed by guest blogger, Kristian Laursen from Cornell University. Site directed mutagenesis...suggests that the final PCR products are pieces of linear, double stranded DNA with homologous ends. These...specified by the arrowheads in (A). Experimental guidelines Primer Design As a rule of thumb, 11 bp of complementary...reaction. Ideally, your primers should be free of palindromic and repetitive sequences, but if present, a minor...the PCR generates a nicked circle rather than a linear product (see figure). Template A high purity plasmid...of the DNA template, and may also decrease the annealing temp of the primers. Because you will be using...

...a000414 (2010). Cleveland Clinic. Gram Stain. https://my.clevelandclinic.org/health/diagnostics/22612-...infected lung tissue, a Danish biologist named Christian Gram realized that certain stains were retained...hardydiagnostics.com/wp-content/uploads/2016/05/Hans-Christian-Gram.pdf Smith, A. & Hussey, M. Gram Stain Protocols...

...PubMed (Link opens in a new window) Gao ZR, Chen WZ, Liu MZ, Chen XJ, Wan L, Zhang XY, Yuan L, Lin JK, Wang...recombinase-expressing mouse lines 2–9 and combinations of transgenic recombinase-expressing animal lines and retro-grade...producing their own INTRSECT plasmids. Online spreadsheet (Link opens in a new window) of Flp-recombinase-expressing...transgenic mouse and rat lines. The Fenno Lab maintains a public folder (Link opens in a new window) of...lateral dorsal striatal cholinergic interneurons. Elife. 7. pii: e39786. PubMed (Link opens in a new window...International 129:104482. PubMed (Link opens in a new window) Poulin JF, Caronia G, Hofer C, Cui Q, Helm... INTRSECT INTRSECT (intronic recombinase sites enabling combinatorial targeting) is a synthetic molecular...

...4. Xue, Lin, et al. "Imaging and manipulating proteins in live cells through covalent labeling." Nature...fluorescent proteins. In-cell labeling strategies The majority of in-cell labeling strategies have two parts... protein (Figure 1f). Labeling strategy pros and cons All of these labeling strategies have trade-offs...Overall, self-labeling tag systems (Figure 1b) are perhaps the best method for live cell labeling and the easiest... biochemists have developed techniques to make labeling chemistry easier and more functional in complex...favorite protein and (2) a synthetic fluorophore-contianing “ligand” that binds to the tag. Like most good... imaging, the initial breakthrough for in-cell labeling techniques was provided by Roger Tsien, who showed...

...good manager? First, it takes an open mind to be willing to learn and grow in skills. Managing a team is...factors that most contribute to job satisfaction: A feeling of being appreciated and valued This often stems... scientists in the work place as described by Sebastiano Massaro in his Nature Biotechnology article “...may not necessarily be comfortable with imposed deadlines (there’s that need for flexibility, sometimes ...

...in DNA. The first, contributed by guest blogger Kristian Laursen from Cornell University, is a bit old ...something great to offer, I just need to get back to emailing (hopefully non-vacationing) scientists. You can...

...ubiquitous, making them a rather blunt instrument for controlling CRISPR. To bring greater control to Acr activity...transfection systems using Agrobacterium (such as Nicotiana benthamiana or Arabidopsis thaliana (Xing et al...of the photoswitchable proteins) fused to mVenus linked to a mitochondrial localization sequence TOM20 ...

... oligonucleotides called morpholinos. When injected into zygotes, morpholinos complementary to a target...features that make zebrafish useful, I’ll outline two recent lineage tracing approaches that led to important...screens. Microscopy-based lineage tracing can help illuminate how signaling pathways—such as those regulated...fish lines have been developed to enable high-resolution mapping of the brain by uniquely labeling neurons...zebrafish have been embraced as a model organism, I’ll outline a few of the available genetic resources and highlight...of the zebrafish genome was published in 2013, revealing that about 70% of human genes have a counterpart...activity and does not work easily in zebrafish.) Morpholinos are a convenient and fast way to deactivate genes...

...quickly along regions calling for abundant, charged tRNAs but will stall at regions calling for low abundance...expressed genes (Emilsson and Kurland, 1990). Controlling gene expression through gene sequence - Proteins...databases showing various organisms’ codon preferences online). Even if the gene is translated, it may not function...synthesis companies such as IDT and GenScript host online codon optimization tools. Keep in mind that, just...and is folding properly. References 1. Angov, Evelina, et al. "Heterologous protein expression is enhanced...PubMed Central PMCID: PMC2970903. 6. Pechmann, Sebastian, and Judith Frydman. "Evolutionary conservation...

...lesioning methods -  they may be the key to further unraveling the mysteries of behavior. We’ve previously ...to the neuronal networks involved in directly controlling muscle activity and coordinating movement, several...stress response circuit by integrating neuronal signalling and triggering release of the stress hormone ...regions such as the nucleus accumbens (NAc), which is linked to the reward circuit, and the medial prefrontal...optogenetics to support these existing findings by controlling neurons in the mPFC with light. Activation of...24882160. Creed, Meaghan, Vincent Jean Pascoli, and Christian Lüscher. "Refining deep brain stimulation to emulate...

...-COMP-b-lac His-tag Cd4d3+4-6his Bio-linker-his Cd4d3-bio-linker-6His* FLAG prey CD200-Cd4d3+4-COMP-blac...Mouse sperm Mouse egg Laboratories using AVEXIS: Christian Sollner, Max Planck Institute Mitch Levesque University...and its resolution by spatiotemporal expression profiling. Martin S, Söllner C, Charoensawan V, Adryan B...

...Typically, regularly spaced denticles (bristles) outline the segments of the Drosophila embryo. In flies... was reading. SHH is involved in the hedgehog signalling pathway, which is a key regulator of all bilateral...1189. PubMed PMID:1905980. Nüsslein-Volhard, Christiane, and Eric Wieschaus. "Mutations affecting segment...

...editing to easily engineer specific transgenic worm lines. The transparent body of the C. elegans hermaphrodite...examples of useful worm CRISPR resources, along with links to protocols, can be found below: SapTrap-SEC kit...Jorgensen Lab. A collection of plasmids that streamlines the production of targeting vectors for tag insertion...from Michael Nonet. Protocol can be found at the (Link opens in a new window) Seydoux lab website . Co-...on miniMos and MosSCI plasmids can be found on Christian Frøkjær-Jensen's Wormbuilder website. General ..., which diminishes epigenetic silencing in the germline. A "FLP-Out" system for controlled gene expression...

...Bogdanove TALEN kit came online in the summer of 2011 and has already become the best-selling kit in Addgene’s... effectors, scientists in several biological disciplines are coming closer to this DNA targeting ideal...arrays from 12-31 repeats. Perhaps one of the most appealing features of the TAL effector arrays is the ease...can be made. Intuitively, one would expect that assembling relatively small, repeat-laden DNA regions into...early in 2011 and they described their kit for assembling TAL effector arrays in a 2012 issue of Nature...Whether you work in a C. elegans lab and have been struggling to mutate a specific gene or your anxiously investigating...constructs for DNA targeting. Cermak T, Doyle EL, Christian M, Wang L, Zhang Y, Schmidt C, Baller JA, Somia...

...Pennsylvania State University. Imagine you are dealing with a defective gene, Xm, the sequence of which...PubMed Central PMCID: PMC5726555. 4. Xie, Kabin, Bastian Minkenberg, and Yinong Yang. "Boosting CRISPR/Cas9...

...expression cell line. Especially if you’re doing a screen. I made a few monoclonal lines (Figure 2) and...control (WT). Vinculin is a loading control. 3. In terms of making the monoclonal line: I had never...cancers) in A549 cells (a human lung cancer cell line), armed only with viruses obtained through Addgene...the edits without any reagent-grade endangered Martian chicory root, but considering this is a needle ...cells were then used to generate a monoclonal cell lines by limiting dilution. The rest of the cells were...indicated multiplicity of infection (MOI). Anti-vinculin Western blotting is a loading control. Pro-Tips... Figure 2: Cas9 expression in monoclonal cell lines generated from A549 cells transduced with lentiCas9...

... in the tissue/which cell type? Location (3): Traveling from the original location - Axonal Transport ...mouse brain (4). An example from the cortex is outlined in the table below. Cell Type Cortex Optimal... the best AAV for your particular experiment! Traveling from the Original Location - Axonal Transport ... (4). Otherwise, using a specific system for controlling gene expression such as Cre-lox, may be sufficient...using Cre-lox, this new method could allow for labeling of neurons showing a very specific phenotype. ...PubMed PMID: 18414476. 3. Aschauer, Dominik F., Sebastian Kreuz, and Simon Rumpel. "Analysis of transduction...

... S., et al. Degron-Based bioPROTACs for Controlling Signaling in CAR T Cells. Cold Spring Harbor Laboratory...variety of constitutive and inducible promoters, linkers, and tags makes EcoFlex a great option for those...and applications.    Plant applications We outlined many synthetic biology MoClo applications, but...protein degradation on chimeric antigen receptor signaling (Kim et al., 2024). The EMMA Toolkit is another...Crossref, https://doi.org/10.1002/tpg2.20312. Brychcy, Merlin, et al. “AspFlex: Molecular Tools to Study Gene... Multiplex Editing: One‐shot Generation of 8× Nicotiana Benthamiana and 12× Arabidopsis Mutants.” The ...

...Since CRISPR is such a robust system, editing or labeling efficiency doesn’t usually change when you add...multiplexing plasmid is available from the Koffas lab. Scaling up your multiplexed gRNAs with Golden Gate and ... overhangs. These overhangs can be customized to link together multiple fragments, allowing ordered assembly...enzyme, the unique flanking overhang sequences can link together to allow for ordered assembly into a destination...overhangs (here, O1-4) adjacent to the cut sites “link” fragments together and drive ordered assembly into...plasmids. Each destination vector contains GFP, enabling you to select cells with high GFP expression. ... separately inducible by L-arabinose and anhydrotetracycline (aTet), respectively. pMAZ-SK contains an...

...what implications their signaling is likely to have). Anterograde tracing outlines neurons from their cell...areas. It is desirable for tracing compounds to outline neurons in the direction of information flow in...terminals of their axons; while retrograde tracing outlines neurons in the opposite direction, from the terminals...so spread across several synaptic connections (Ugolini et al., 1989). When using these viruses to mark...neuronal tracing has the added benefit of only labelling the subset of infected cells that also express.... Differences between AAV serotypes may be also linked to differences in viral uptake by cells and to ...identification requires both antero- and retrograde labelling, synaptic restriction as well as controlled monosynaptic...