... of Antonello Bonci, Director of the National Institutes of Drug Abuse Intramural Research Program (NIH-NIDA... I work in), an obvious behavioral experiment to test for sufficiency of your circuit of interest in promoting..., on the other hand, would not be an appropriate test.  Of course, proper controls are required and include...sutures and subsequent determination of the proper coordinates of your brain region of interest (as shown in...identical for all of these surgeries. Identify coordinates through a rodent brain atlas or research papers...prior to using the virus. You may need to adjust coordinates based on your own method of identifying bregma...readout. The advantage is you can “zero” your coordinates at bregma which allows you to quickly and reliably...

...regularly, and mix samples very well.  Use duplicates or triplicates to increase the accuracy of your experiment...viral vector production facility, food allergy testing lab, and the grad student down the hall from you...-fold dilution series. Each step of the series dilutes by 1:2 for a series that ranges from 1:2–1:32. ...standard dilution series in duplicate or triplicate. Replicates allows you to calculate the curve’s coefficient...replicate measurement divided by the mean of all the replicates multiplied by 100. To calculate the %CV of the...ideal %CV is less than 15%. You should also run replicates for all of the samples in the assay, as this ...these.  Review previous literature or results from labmates to get a sense of the concentrations you might...

...CNS cell types, including microglia, astrocytes, and oligodendrocytes, provided that reporter genes specific...activators to induce gene transcription at genomic sites targeted by specific sgRNAs. Its RNA-guided nature...packaging limits (i.e., how many sgRNA expression cassettes will fit). Cre-dependent expression of the SPH...selectively target these different transcriptional start sites in order to better understand isoform-specific gene...activity (hence, “immediate-early”) and consequently regulates neuronal physiology by promoting the development...gene possesses 9 distinct transcriptional start sites, all of which yield the same BDNF protein. To demonstrate...cell types (microglia are a notable exception) necessitates such molecular gymnastics for sensitive applications...

...called a Kirby-Bauer disk diffusion test or Kirby-Bauer susceptibility test (Figure 2), and requires at least...shows how to perform a Kirby-Bauer disk diffusion test and check bacteria for antibiotic resistance. Source...bacteria are causing the infection before they can test for resistance. Growing and isolating the infecting...infecting bacteria can take 24 hours or more, and testing for resistance often takes an additional 18-24 hours...available. For routine infections, like strep throat, testing for antibiotic resistance isn’t commonly performed...infections is so rare. When antibiotic resistance testing is requested clinically, this involves growing ... treated. We can prescribe antibiotics for the shortest time period possible to effectively manage a patient...

... bacterial ARG from Serratia sp. 39006. We next tested bARGSer with several inducible expression systems.../L yeast extract, and 5 g/L NaCl) or on LB-agar plates containing glucose and L-arabinose at 37oC for ... arabinose and colonies on arabinose-containing plates will be visibly white, and gas vesicles in cells...from left to right: colony whiteness on inducer plates, white inclusions in cells with GVs via phase contrast...mARGs also had a large genetic footprint (two cassettes + a booster cassette), their expression heterogeneity...mARGs come as a set of two PiggyBac integratable cassettes, both with doxycycline inducible promoters (Fig...increased ultrasound contrast. In all the cell lines we tested, 1 µg/mL doxycycline was sufficient to saturate...

... order fulfilment from an average of 40 minutes to 10 minutes. While users can’t tangibly see these improvements...each project. This creates accountability on the part of each developer and creates trust amongst the ...collaborate with the Product Team who in turn collaborates with stakeholders to resolve the questions.  ...would be staying on top of any third party library updates. Third party libraries are reusable software components...with their third party libraries and any security updates. Most of the time, updating these libraries is ...this year, which means there will be no further updates, security or otherwise. This might sound like Greek...

...restriction sites flanking the promoter-gRNA construct, but with different sequences adjacent to the sites. When...genome by removing the sequence between two target sites. Modifying multiple genes at once. Using multiplexed...promoter-gRNA constructs in order to target multiple sites! In this post, we'll cover Cas9 multiplexing options...oligonucleotides. These plasmids contain Type IIS restriction sites that flank the promoter-gRNA constructs. When these...unique overhangs (here, O1-4) adjacent to the cut sites “link” fragments together and drive ordered assembly...transcribed together and are flanked by specific sites that allow them to be cleaved and released. These...smaller than constructs with multiple promoter-gRNA cassettes, making them advantageous for small capacity vectors...

...personal litmus test was whether my wife could explain what I was proposing. The result of my test was humbling... your candidacy. A major reason that a lot of candidates either get no interviews, or get several interviews...accomplish. No matter how many times I say this, candidates regularly try to convince me that their search...language as possible. Seriously. My suggested litmus test is whether your friend in a lab outside of your ... and therapies." This is only the first few minutes of the chalk talk. Realize that it is rare to make...available here for free!) contains strategies, hints, anecdotes, and insights. After I wrote the first version... Jennifer Lippincott-Schwartz at the National Institutes of Health, was an Associate Professor at Albert...

...DNA with a TA dinucleotide (there are many such sites in the human genome). The original plasmid is empty... then degraded by the cell.4.) The transposase creates a double strand break in the DNA and allows the...has been designed to perform gene transfer in vertebrates. The system is comprised of 2 components: 1) ...number of extinct transposon sequences and then testing various combinations of changes. The basic functional.... The vectors each contain two SfiI restriction sites with different overhangs depending upon whether ... Feb 4. doi: 10.1002/biot.201400821. PubMed. 2. Mates L, et al. Molecular evolution of a novel hyperactive...transposase enables robust stable gene transfer in vertebrates. Nat Genet. 2009 Jun;41(6):753-61. Epub 2009 ...

... incubation (10–20 minutes) at 70 °C, or a truly lengthy incubation (30–60 minutes) at 37 °C, if you suspect...DNA-binding proteins, you may need to sonicate your lysates in order to release the proteins from their binding...in your background, try asking more experienced labmates or looking it up online. You’ll find many resources...your timer to ding. If you’re shaving off a few minutes here and there, and seeing high background in your...antibodies). You’ll want to run a protein gradient to test the antibody at the recommended dilution. If the...concentration decreases).     Figure 3: Testing the antibody’s dynamic range. A) The protein dilutions...from 1:100–1:1,000. If your antibodies come from ascites fluid, try a dilution range from 1:1,000–1:100,...

...your incubation time on ice to 5 minutes (incubating the full 30 minutes will not significantly improve ...standard primers (no need to add unique restriction sites on the ends) and amplify your sequence of interest...the PCR product and TOPO Vector.  3. Incubate 5 Minutes at Room Temperature: You can place your reaction...digest, or sequencing. Pro tips Do not add 5’ phosphates to your PCR primers; you need that free hydroxyl...functionality are used in place of Taq to reduce error rates; however, proofreading polymerases will also remove...); however, you may need to incubate for 20-30 minutes if your PCR product is at a low concentration or...

...growth and DNA yield. Test multiple, small colonies After streaking LB agar plates, you may notice that...vectors (LTRs and ITRs), plasmids with multiple polyA sites, and other plasmids that contain repeating elements...how to amplify recombination-prone plasmids and testing your prep can save you the headache of troubleshooting...cultures contain the recombined vector backbone. Test multiple colonies before saving a glycerol stock...you can try re-streaking for single colonies and testing multiple preps. In some cases, we suggest gel extraction...the recombined plasmid is still visible. This indicates that the DNA prep contains a mixture of the full...

...for the targeted gene. After initial testing, Dickinson et al. tested this system across 7 genes in parallel...remove after injection, they added flanking loxP sites and a heat-shock inducible Cre to generate a self-excising...worms have laid eggs, hygromycin is added to the plates to select for candidate knock-ins. These candidate... is inserted at the 5’ end of a gene, the SEC separates the targeted gene from its promoter, creating ...protein in the C. elegans genome. Although they tested fluorescent protein fusions, this system could ...editing using linear DNAs with microhomology to Cas9 Sites in Caenorhabditis elegans. Paix A, Wang Y, Smith...

.... It’s a problem that he considers “one of the greatest challenges of neuroscience.”  Hot plasmids: Twitch...inside cells that are on the move, such as T lymphocytes. Griesbeck and his colleagues began by isolating...version of the protein with as few calcium-binding sites as possible for added sensitivity. As reported in...these Twitch sensor variants were optimized by testing various linker configurations in a large-scale ...and high resolution functional tracking of T lymphocytes, too, making them a versatile tool for application...other people start using it. For us, it’s like a test.” References Thestrup et al. Optimized ratiometric... functional in vivo imaging of neurons and T lymphocytes. Nat Methods. 2014 Jan 5. doi: 10.1038/nmeth.2773...

...research tools. BenchSci collates information about antibodies while FPbase curates information about fluorescent...sometimes formulating new biological concepts. However, rates of reproducibility for preclinical studies, as reported...Project: Cancer Biology have produced similarly low rates of reproducibility. Failed attempts to apply irreproducible... to future work cost both time and money with estimates reaching in the billions of dollars (Freedman ...making it easier to retain searchable, thorough notes on how lab procedures were actually performed, potentially... The publication record will then show that the tested hypothesis is correct (a great success story) even...category include performing experiments with too few replicates leading to suspect statistical significance (...

...reinhardtii. When exposed to blue light, this channel activates neuronal activity by opening and allowing positively...conformational change in the extracellular domain which activates the native signaling cascade of the intracellular...conformational change when exposed to blue-light and activates downstream secondary signaling pathways. Photoswitchable...light, the ligand engages the binding pocket and activates the receptor. Created with BioRender.com. ...factors will help you determine which one(s) are candidates for your experiments.  Wavelengths Most vectors...et al., 2019) for each tool, and you may have to test and optimize the intensities of light sources used... frequencies (Zhang & Cui, 2015). You’ll need to test for these differences when designing your experiment...

...Light-induced cell ablation with MG-2I-dL5** He et al tested out light-induced cell ablation with MG-2I-dL5*...dye and exposed to light stained dead within 30 minutes of exposure. 90 seconds of illumination were sufficient... fish with the dye. Embryos exposed to just 12 minutes of illumination had fewer mCer3-labeled cells in...cells.  He et al also show that MG-2I-dL5** only generates small amounts of non-specific ROS when exposed... red fluorescent photosensitizer protein that generates ROS when illuminated with green light. They found...that KillerRed required longer exposure times (5 minutes vs 10 seconds) to achieve the same level of killing...cell nuclei. The two photosensitizers had similar rates of off-target damage to neighboring cells. TL;DR...

...nuclear processes. The latest of these visualization tools is CRISPR-Sirius, the brightest CRISPR-based tool...CRISPRainbow allows for simultaneous visualization of sites on different chromosomes in living cells, the system...PP7, or boxB sequences inserted into the sgRNA generates blue, green, or red colors for labeling. A combination...in CRISPRainbow, they knew they could be good candidates for further development. They inserted an octet...three loci and measure inter-locus distances. They tested this system out on chromosome 19 by targeting 46... the National Academy of Sciences of the United States of America 112 10 (2015): 3002-7. PubMed PMID: ...

...collection of pathogenic Klebsiella and Pseudomonas isolates causing urinary tract infections. This project... for plant compounds to treat complications of diabetes Gloria Otito Izu, PhD Candidate, University of... the major pathophysiological complications of diabetes mellitus. Recombinant human proteins that play...play a role in insulin regulation and diabetes will be expressed from plasmids (find more information on...of the plant compounds on these proteins will be tested. Investigating changes in neuronal activity after...Armenia Gohar Tsakanova is a Senior Researcher who investigates the molecular and cellular etiopathomechanisms...projects progress. Subscribe to the Addgene blog for updates and future collaborations. Many thanks to all of...

... an entire experiment and delegate tasks to lab mates or to discuss a potential new research direction... regular check-ins with their employees. 15-30 minutes weekly or monthly will usually suffice to keep ...Managers will get a sense of the employee’s workload, updates on current projects, and offer feedback. The employee...anything that is missing.  During the meeting, take notes on the agenda points. At Addgene, we like to do ...Before leaving the meeting, skim back through your notes and pick out the action items. Move them into a ... positive peer pressure to make sure everyone completes their assigned items to keep the project moving...scope” above) or if you can cut it down to thirty minutes. Keep it moving: If you tend to get stuck diving...