...the blocking-only templates will compete with the blocking/intended mutation templates resulting in cells...Ludwig-Maximilians-University in Munich and Marc Tessier-Lavigne’s lab at the Rockefeller University in ...have focused on strategies to improve overall HDR rates, we have shown recently that desired genome-editing...unwanted indels. Following this observation, we tested a strategy that was discussed in the field at the...described project while he was a Postdoc in Marc Tessier-Lavigne’s lab at the Rockefeller University in ... NYC.   Dylan Kwart is a graduate student Marc Tessier-Lavigne’s lab at the Rockefeller University. He...

...start defining keywords you’ll want to find in candidates’ resumes or CVs; “vivarium”, “weaning”, “colony...will help save time by screening out irrelevant candidates, and, more importantly, it’ll help you define...personnel salary ranges, go to online job information sites (like, indeed.com, or salary.com). Search industry...is important to ensure that you’re looking at institutes that have the means to support your work. For...work requires specialized equipment, try finding institutes that offer shared equipment that you can use ...the anxiety that comes with negotiations. This creates a self-assured discussion when it comes time to...faculty position because none of the offering institutes were able to provide the resources required for...

... viruses. Now focusing on plant biology, she coordinates the Network for the Genetic Improvement of Cowpea...agricultural plant. Her current research focuses on endophytes, bacteria, and fungi that reside within living...proteins and enzymes. These compounds will later get tested for their potential agricultural and medical use...detect glyphosate via H2O2, one of the reaction’s metabolites. One useful genetically encoded H2O2 probe is...projects progress. Subscribe to the Addgene blog for updates and future collaborations. Many thanks to all of...References 1. Martinez-Klimova, Elena et al. “Endophytes as sources of antibiotic” Biochemical Pharmacology...

...analyze sequence tool: Annotates features, BLASTs, aligns, digests, and translates a sequence.   NEBCutter2...restriction enzyme cut sites Webcutter 2.0: For finding restriction enzyme cut sites Open reading frame finder...sequences in a database ExPASy translation tool: Translates a DNA sequence using all six open reading frames...primer design tools, but here are a few stand-alone sites for general primer design, as well as primer design...

...separate resistance cassettes and your gene of interest is cloned into/inactivates or completely removes...translocation Bacteriostatic 5-25 ug/mL in EtOH *In prokaryotes.     **Dissolve in dH2O and sterile filter unless... are trying to disrupt or delete, which both inactivates the gene and acts as a marker for the mutation...months. Ampicillin breaks down especially fast and plates should be used witin 1 month for optimal efficiency... them to media hotter than about 55oC and store plates/stocks wrapped in foil if a light-sensitive antibiotic...

...mentorship is by recruiting your own peers and classmates! Your mentors don’t need to be more senior or...that there’s some correct level of intimacy that creates the magic of a safe space. No matter why, now is...roles but for almost every position, we look for candidates that have demonstrated initiative. In fact, hear...opportunity to act or take charge before others do”. Candidates who have “started something” which they can share...have a definite advantage in our process. We see candidates who have done the following types of things: ... a character count in your head.  Here are some sites that have compiled lists of grants: Science: ...

...“PCR test” has recently transitioned from jargon to non-jargon in the context of disease testing, it can...other uses. Many laypeople would use “PCR test” to mean “a test that uses a biological sample to look for...presence of a virus.” A scientist would likely mean “a test for the presence or absence of a predefined DNA ...Science to Elementary and Middle Schools   Image courtesy of Ron Mader, via Flickr, under the Creative Commons...

... scores. That player doesn’t trust his teammates; the teammates don’t trust him. This team is not going...includes: Michelle, the Senior Scientist who coordinates all kit creation and quality control efforts ...informed decision about something? Fix a problem? Test a hypothesis? Whether you officially call it a “...I’m an introvert!” is not an excuse. Every psych test I’ve ever taken has declared me an introvert, and...academic labs, uses Slack constantly. Whether your teammates are two desks away or on the other side of the...perfectly clear lines of communication, but if teammates don’t trust each other, none of that matters. ...

...way to re-create a functional virus. Indeed, by testing several vectors and packaging cell line combinations...proto-oncogenes or with human samples that have not been tested for HIV.  If not using proper technique, there’... from the LTRs) has been removed, Rev (which facilitates nuclear export) is expressed from a separate ...by your transfer vector) binds to the TRE and activates the expression of your gene. The addition of Dox...virus precipitation. My tip for this process is to test several protocols that have been described (protocol...viral capsid antigen p24, the FACS dosing method correlates viral titre to the number of cells transduced...ratio of virus to cells). Again, my tip here is to test several protocols as the set-up depends on the type...

...Surveyor assay to look for genomic edits at the BRAF sites targeted by my specific gRNAs (Figure 4). The most...the 428 bp Surveyor nuclease cleavage product indicates a gene edit. GFP gRNA-transduced Cas9-A549 cells...cleavage products. The appearance of the 428 bp band indicates edits in the BRAF gene in the BRAF-gRNA transduced... product. Figure 5: gRNA-targeted BRAF sites were edited in A549 cells. Surveyor nuclease was...those formed from control (GFP gRNA-targeted) BRAF sites (lanes 3, 4) were used as controls. Duplexes were...a gene knockout was straightforward, and is a testament to how far genome engineering has come. For comparison...Population by Limiting Dilution Lentivirus Production Websites Primer3: for Designing Primers UCSC Genome Browser...

...broth and on the agar plates. Mycoplasma colonies are easily visible on the agar plates. This method is capable... to poor lab techniques. These include reusing pipettes for multiple cell lines rather than using disposable...always use a second assay to be 100% sure. Before testing for mycoplasma, the cells should be in continuous... for low levels of contaminations to grow. For testing, the medium should not have been changed for at... (Uphoff and Drexler, 2011). Culturing on agar plates/broth is considered the ‘gold standard’ for detecting... are tightly packed with seals. Use disposable pipettes to avoid cross-contamination. Make sure you keep...

...and not exposed to CO2/dry ice. All samples were tested in duplicate. What we found Not all tubes (or ...) were permeable to CO2 at low temperatures. We tested two different types of tubes: the ones we use to...shown). What we did next: Venting the tubes We then tested whether venting the tubes prior to thaw released...What about putting it back in the freezer? We then tested whether returning the tubes to the -80°C freezer...would prevent the pH change upon thaw (Fig.4). To test this, we froze the tubes at -80°C and then moved...affects AAV function. Furthermore, another study tested AAV infectivity after a range of pH exposures. ...

...majority of research institutes.  Pro tip! In most academic and nonprofit institutes, the Tech Transfer ... signatures from two different institutes. Our online portal automates a lot of the process, from notifications...  An MTA is simply a contract or agreement that states the terms of use for a tangible research material...people think our MTAs are different than other institutes. But they’re not! The vast majority of our plasmids...

...the reporter mRNA, which contains the tethering sites for the fluorescent proteins and is driven by an... copies of the PP7 and MS2 coat protein binding sites to tether many copies of the FP-coat protein fusions...NLS-PCP-GFP is removed by the first ribosome that translates the mRNA. To permit tracking over time, Yan et...Each time the reporter is translated, the SunTag sites on the nascent polypeptide recruit scFV-GFP, generating...translation in various cell types and physiological states. References 1. Ingolia, Nicholas T., et al. “Genome-wide...SunTag Catch up on Your GFP Background Choose the Brightest Fluorescent Protein Resources on Addgene.org ...

...yield, and when transiently expressed in cells, generates much less signal than comparable fluorescent proteins... very similar to the widely used FRET, but it eliminates the need for an excitation light source. Instead...multiple subcellular compartments over several minutes using Nano-lantern fusion proteins. To further ...continuously imaged cells for about 4 hours! The latest enhanced Nano-lanterns (eNL) described in Suzuki...fluorescent imaging! These eNL constructs use the brightest characterized luciferase, NanoLuc, and its substrate.... Filtering the emission wavelength partially separates the signals of various Nano-lanterns, but it does...

...Why do scientists use yeast vectors? Yeast are eukaryotes and thus contain complex internal cell structures...technical advantages that come with working with prokaryotes. This includes but is not limited to: rapid growth... selection when working with yeast, due to high rates of spontaneously occuring resistant mutants and ...complementation is weak. yes   LYS2 L-lysine yes - Grow on plates containing alpha-aminoadipate in the absence of...physiological levels due to high copy numbers. This creates a potential metabolic burden on the yeast cells...markers. The MX series of antibiotic resistance cassettes is most commonly employed, with the KanMX being...

...microscopy, a metal disk containing a number of holes rotates through the excitation light path. Each hole corresponds...happen.Using red light has a second advantage: red light penetrates deeper into biological tissue. To prove this,... create a thin sheet of excitation light that propagates perpendicular to an imaging objective that collects...into the sample via an evanescent wave that only excites fluorophores within a few hundred nanometers of...Fluorescence (TIRF) microscope can be used. TIRF only excites fluorophores within a few hundred nanometers of... pixel by pixel are unable to provide the frame-rates necessary for imaging tissues of this size. For ...Fluorescent Proteins for Multi-Color Imaging Choose the Brightest Fluorescent Protein for Your Experiments Resources...

...autoinflammatory loop: keratinocytes both respond to and release IL-36 cytokines. Keratinocytes are the main source...cytokine family resulting in the activation of keratinocytes, which release inflammatory mediators that trigger...known how IL-36 cytokines actually get out of keratinocytes and induce their effector functions. It’s an...important to study what is happening within the keratinocytes. “We hope to use CRISPR libraries from Addgene...or proteins are involved in IL-36 release in keratinocytes,” says Mellett. This would help the lab identify...

...’s not, I can always take more time to check my notes!  Know your purpose The purpose of our assignment...assignment was clear: present a paper to our classmates so they could understand it. When my mind went blank,...gene of interest incorrectly on slides 8-16. Your notes disappear. The wrong version of your slide deck ...simple as telling yourself, “I can stop and check my notes if I need to,” or “It is okay if it doesn’t go perfectly... field, with very little text on the slides and notes instead of written speeches. When you’re doing all...speak at conferences, or even just provide quick updates during a company meeting increases visibility of...

...and MEK1 is active or “on.” Dronpa’s color thus indicates MEK1’s current state (Green when off, dark when...photoswitchable Dronpa domains were inserted at sites homologous to the locations they were attached to... a few examples demonstrated in Zhou et al. 1. Testing kinase inhibitors Zhou et al examined the ability...indicator of MEK1 activity. When MEK1 is active, it phosphorylates the ERK KTR and red fluorescence localizes ... fluorescence can be found in the nucleus. For testing kinase inhibitors, psMEK1 and ERK KTR-mRuby2 expressing...blot. Phosphorylation of MEK1/2 peaks at just 5 minutes post-Raf1 inactivation, and then decreases. Treatment...