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...scientists are acutely aware of these problems that directly affect their daily lives and future prospects....structure of the biomedical workforce. Quotes taken directly from participants (summarized in 2) touched upon...information on number of postdocs” “Postdocs are really hired to produce results, not scientists”  These comments.... Melanie Sinche is a STEM career counselor, Director of Education for the Jackson Laboratory for Genomic... at Scientific Meetings from Addgene Executive Director Joanne Kamens Learn how to be a successful manager...

...’ve got you covered. Defining your control requirements To pick the best tool for the job, it’s important...important to know what the job requirements are. Here are the questions you should be asking. What kind ...recombinase. This is a time-tested system but does require knock-in of the LoxP sites, which is more difficult...control all the time If your experimental system requires tunable control - or maybe you’re just a control...time consuming. If your native protein doesn’t require this level of control, one of the previous options...

... viral particles. Direct measurement of lentiviral RNA is one alternative to direct p24 measurement. Like...The two most common assays for physical titer are direct p24 measurements via ELISA and qPCR for viral RNA.... Developing the proper control line typically requires transducing with a limiting multiplicity of infection...labs that will routinely be producing virus or require precise titers. Additional considerations for titering...

...left school when she was hired full time as a Lab Technician. “I was so tired of being in school so I ...student and I truly enjoyed learning.” Addgene’s Director of Product Management Eric Perkins, who has been... was devastating. At the same time, she has an entirely new path to discover and gives her time to give...interviewees. Fabienne Furt, Addgene’s Associate Director of Business Development, got her PhD in Plant ...

... allele decreases. Crossing mice to generate a desired phenotype Instead, genetic engineering should be...in the journey towards producing mice with your desired combination of mutations. Assuming your parental...generate mice that are homozygous for all of your desired genes. Remember Punnett squares from high school... are homozygous (or hemizygous) at all of your desired alleles to conduct your experiments. You should...target gene in your experimental mice. This will require several rounds of breeding in which you’ll want...

...reviews our sequencing QC standards to ensure the entire team is using the same rubric and making the same...process also highlights other QC topics that may require additional attention during analysis. These include...and nucleotide populations. All of these topics require additional considerations to resolve. Sequencing...help QC scientists determine if enough reads were acquired from the MiSeq for assembly or with enough coverage...nanopore sequencing. This platform sequences the entire plasmid in one read and thus can resolve sequencing...

... genome editing for a few months. More time is required for its optimization and development before it...features of NgAgo First, unlike Cas9, NgAgo does not require a PAM sequence for target recognition, which gives...Additionally, in vitro assembly of NgAgo/ssDNA requires incubation at 55 °C - a dangerous, non-physiological...regions of the genome or just after a stop codon (as required for C-terminus epitope tagging). NgAgo survey ...respondents "Final Verdict", is that more time is required for NgAgo optimization before we can make any ...

... a plasmid whose copy number is unknown, it is entirely possible that you are working with a low copy ...Start with a single, fresh colony Sub-culturing directly from a frozen glycerol stock or agar stab may ...exchange will prevent your cultures from reaching the desired density. The volume of your flask or culture tube...cultures, especially those in deep-well plates, require a faster shaking speed for proper aeration (260... temperature. These recommendations also often require a longer growth time. Note: Julian Taylor-Parker...

...end-joining (NHEJ) to make deletions or homology-directed repair (HDR) to create precise genome edits. But...end-joining)? MMEJ, a form of alternative end-joining, requires only very small homology regions (5-25 bp) for...spent cloning. In contrast, the short homology required by MMEJ can easily be added via PCR amplification...genomic DNA. These three double stranded breaks are repaired by two rounds of MMEJ. This double MMEJ strategy...Resources on the Addgene Blog Brush up on Homology Directed Repair and Non-homologous End Joining Read About...

...light only kills microorganisms that it strikes directly. UV light exposure is also harmful for eyes and...around your work area. Make sure to only bring required materials into the hood. This will give you enough... avoid sticking pipettes into solution bottles directly, rather decant the solutions into disposable sterile...depending on the cell line so old cells should be retired! Conversely, cells younger than the 3rd – 4th passage...blogger, Sana Khan Khilji! Sana Khan Khilji recently acquired her Master’s degree in Biochemistry from Freie...

...cell, a minimum of 2 kb of sequence homology is required, but 6 to 14 kb of homology is typical for targeting...conditional knockout. This experimental design requires two rounds of recombination – the first removes... recombination can occur in multiple ways. The desired recombination event will remove NeoR only and leave...genomic sequence and create a break that can be repaired by homologous recombination using a repair template...on the Addgene Blog Get the Basics of Homology Directed Repair Read about Generating Knockouts with CRISPR...

.... In this event, the degron domain shuttles the entire tagged protein to the proteasome for degradation... additional sequence (7 kDa). This system also requires less genetic engineering than some of the other...other systems reviewed here. The only required components are 1) a SMASh-tagged protein and 2) asunaprevir.... An obvious drawback of the system is that it requires an additional component to be engineered or expressed... with additional components beyond the minimum required for a degron system, then it’s probably best to...

...a few aspects in which spectral flow cytometry requires special considerations. Conventional flow cytometry... your panel. Highly expressed markers should be paired with dimmer dyes so they don’t “wash out” the rest...Conversely, markers with low expression should be paired with brighter dyes, so they can more easily be ... two overlapping cellular markers should not be paired with these dyes, as this would create difficulty...samples stained with a single fluorophore. They’re required for every fluorophore you use in your panel, whether...

...stripes in the acquired image (5).  A way to get around this problem is to illuminate and acquire images of... of the solitary tract.  The arrows point the direction of the specimen, A= anterior, P= posterior, D=...  Furthermore, point-by-point scanning along the entire specimen makes imaging too slow and unwieldy for...) sensors, it takes only a few milliseconds to acquire one image, thereby significantly reducing the time...

...AAV replication uses the host’s polymerase, but requires Rep proteins to process a double-stranded intermediate...work with. These plasmids contain ITRs which are required for proper packaging of AAV, but also form secondary...AAV than I think I need. Unlike lentivirus, AAV requires a higher number of viral particles for efficient...several hours on a heated stir plate to dissolve and requires monitoring so that the solution doesn’t get too...AAV for my cells of interest. Every batch of AAV requires its own MOI optimization to account for batch-...

...transfer plasmids have a wildtype 5'LTR, which requires the presence of the Tat protein to work, while...'LTR. Including a chimeric 5'LTR removes the requirement for the HIV Tat protein, thus decreasing the ...gag, pol, rev, and tat. 3rd generation systems require an envelope plasmid, and two packaging plasmids...2nd-generation system because the wildtype 5'LTR promoter requires Tat to function. Third-generation plasmids can...plasmid(s) contain the minimal number of HIV genes required for virus production (3 or 4). Third-generation...

...bacterium, and is essentially always there, or acquired, which means the bacterium is not naturally resistant...intrinsically resistant to this class of antibiotic. Acquired resistance can be the result of one bacterium ... died for instance). These three mechanisms of acquired drug resistance are referred to as methods of ... to a drug while a full course of treatment is required to wipe out those bacteria that are partially ...Kirby-Bauer susceptibility test (Figure 2), and requires at least 18-24 hours to obtain results. While ...

...contamination: Epithelial Mesenchymal Transition Is Required for Acquisition of Anoikis Resistance and Metastatic...from the official biorepositories or have been directly isolated from patients. In these cases, a test...outlined in this article. Now many journals strictly require evidence of cell line provenance and identity. ...Council (BBSRC) and Medical Research Council (MRC) require authentic cells to be used and STR profiling to...can be prevented and we can progress faster to desired clinical endpoints.   Nick Amiss is an ATCC Outreach...

...created a yeast-based directed evolution approach with some notable improvements. Directed evolution techniques... fluorescent protein--no affinity purification required! The team used other molecular techniques to introduce...microtubuli and plasmids tagged with MapTau to label entire microtubules. Find these fluorescent mammalian ...contains the signal peptide of MBP, followed by the desired nanobody (NB) sequence, an AviTAG, and 6xHis tag...

... by collections of neurons that are intricately wired into circuits through synaptic connections (Shepherd...cross-react with other antibodies, or are unavailable entirely. The other advantage is that these tags are small...intrinsic visual signal, they cannot be detected directly in live imaging experiments. It is therefore difficult... in the brain and can be detected through their direct fluorescent signals or using anti-FP antibodies...primary antibodies which can be used to amplify the direct smFP signal within sub-cellular structures. For...

...incredible versatility opens aptamers for use in many entirely new applications including aptamer based fluorescent...sensitive detection assay. In 1999, Grate and Wilson paired laser cleavage and an RNA aptamer that bound the...to induce fluorescence outside the context of a paired fluorophore protein. Years later, Samie Jaffrey...mimicking GFP.  Though the aptamer, called Spinach, required longer exposure time than its protein equivalent... less immunogenic than proteins. Assays that require recognition of ions or particularly small molecules...

...tools, approaches, and infrastructure necessary for direct tests of cause and effect hypotheses between gene...unavailable.” As stated by one of the NSF program directors, this program should “enable the ability to study... in the tree of life.” Accepted proposals were required to present research plans that went beyond obtaining...is no small task. The EDGE program solicitation requires funded labs to rapidly disseminate protocols, ...microscope with polarizing filter. Development is direct in this species, and hatchlings are around 1mm ...

...in different CGBE tools allow the researcher to direct the repair of the abasic site. But what if an editor...transversion editors. Red arrows indicate a conversion directly catalyzed by the base editor. Black arrows indicate...this point relied on deaminase enzymes, either to directly accomplish the base edit or to create the substrate...they realized that MPG could be engineered to act directly on unmodified G bases, resulting in a G → Y (Y...Conclusion With these advances, every base can now be directly targeted by a base editor, although the efficiency...

...Historically, molecular interactions were detected by indirect means often using probes with the potential to...proteins, pioneers of this technique were able to directly detect protein/protein interactions in living ... to each other in the spectrum, you can easily directly excite the acceptor with the laser used to excite...corrective approaches and appropriate controls are required to make this method useful for dynamic experiments...fluorescence, this method is also less sensitive to direct acceptor excitation artifacts and it is possible...

...). These so called “plasmid addiction systems” require dividing cells to retain plasmids - if a cell does...Figure 2). This MazF-ZeoR cassette is flanked by two direct repeats which can be used for marker removal so..., knock in a GFP cassette, and introduced site-directed mutations, all without introducing unwanted selection...short-lived RNA antitoxin; Modulation of killing (mok), required for hok translation. Here, Mok and Hok translation...Sok antisense RNA regulates hok gene expression indirectly through the overlapping mok gene." Journal of...

...system (OTS) for the incorporation of phosphoserine directly into recombinant proteins (5). Optimization of...of this system defined three components required for translation (Figure 1): A tRNA that recognizes a... The generation of pSer-incorporated proteins requires a molecular toolkit containing the OTS, an appropriate...phosphoserine OTS, Sep-OTS, contains the components required for the translation of pSer-incorporated proteins...phosphoserine must be recoded to ‘TGA’ using site-directed mutagenesis. In the C321.ΔA strain, this TGA will...

...a stupendous listener, but I aspire to  be. If you don’t at least aspire to be a good listener and consciously...each other disagree about a specific decision, direction, or goal. Resolving these types of conflicts is...Assumptions: Making incorrect assumptions can be a direct cause or consequence of lapsed communication. Making...job”. It’s certainly resolvable, but will likely require a manager to explicitly assign the responsibility...

...3 days to complete the entire experiment, but you’ll have to spend the entirety of each day doing the ...work in a job where you’re pulled in a thousand directions at the start of the day, writing your to-do list...depositors from Boston University Contact Dr. Gilmore directly to see if he knows anyone who’s interested in ...if I had planned my day extensively, my lack of desire to go sit in the tissue culture hood may have prevented...

...interest (Fire et al., 1998). The discovery of RNAi in the worm actually rewarded depositors Andrew Fire and...Yeast are also the first eukaryotic genome to be entirely sequenced and is very amenable to genetic manipulation...utilized the worm to study calcium signaling in the entire adult brain, using a plasmid that expresses a genetically...(1974): 71-94. PubMed Central PMCID: PMC1213120. Fire, Andrew, et al. "Potent and specific genetic interference...

...instability at 37°C (Ouellet et al., 2016) and requires a tRNA scaffold to improve its stability in vivo...Ming Hammond’s group created Spinach2 using site-directed mutagenesis of Spinach. In doing so, they identified...green fluorescent aptamer, was identified through directed evolution. First, a pool of RNAs that activate...Fluorescence is observed when these RNAs complex and the entire aptamer can hybridize and fold. (Alam et al., 2017...fluorescent protein by fluorescence-based selection and directed evolution." Journal of the American Chemical Society...