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TypeBlog Post...can rapidly and efficiently evaluate Cas9 cutting sites and activity (Knudsen et al., 2018). To create ...lab compared editing at three different genomic sites in the AAVS1 locus using GEmCherry2. In this experiment...again that sgRNA structure and genomic recognition sites play a large role in CRISPR-Cas9 efficiency. They...
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Hot Plasmids - May 2022
TypeBlog Post... both on and off target. Across all loci tested, error rates overall ranged from 0.3% to 10%, in comparison...Importantly, this is true in both mice and nonhuman primates (for AAV.CAP-B10, for example). Given that the... -
Flow Cytometry Readouts: Yes, No, and Everything in Between
TypeBlog Post...to the right indicates a higher signal, or upregulation, and a peak to the left indicates a lower signal...continue gating populations of interest with normal gates, just based on a histogram plot instead of a dot... -
Viral Vectors 101: Gamma-Retroviral Packaging Systems
TypeBlog Post... packaging cell line. This system provides the greatest flexibility to pseudotype γ-retrovirus using different...post‐transcriptional regulatory element; sequence that stimulates the expression of transgenes via increased nuclear...U5 and the rest of the provirus. 3’ LTR Terminates transcription started by 5' LTR by the addition... -
Antibodies 101: Flow Cytometry Controls
TypeBlog Post...companies or check the literature. Pro tip! Early gates, like your live/dead markers or commonly used markers...specific cell populations, often used in the later gates, are more likely to require FMOs. Isotypes Isotype...rather than for setting up your initial voltages and gates. Ashlyn has a research background in CD8+ T cells... -
CasPEDIA: A Functional Classification of Cas Enzymes
TypeBlog Post... trans activity. AsCas12a, on the other hand, generates staggered DNA nicks at its target in cis and cleaves... used by Jinek et al. 2012, or browsing for the latest AsCas12a plasmids, Addgene and CasPEDIA have got...from CasPEDIA. How can I use it? CasPEDIA operates like many search engines, featuring both a general... -
Viral Vectors 101: Viral Applications
TypeBlog Post...be used to express proteins. Viral expression cassettes typically contain a promoter (inducible or constitutive...lineage, and so much more! Libraries allow you to test thousands of elements simultaneously in your system...dose. Vaccine development strategies. Image courtesy of Gorry et. al, 2007. Clinical applications... -
Opto-Nanobodies: Using Light to Manipulate Cell Signaling and Protein Purification
TypeBlog Post... start, the team inserted AsLOV2 into conserved sites in all eight surface-exposed loops of an mCherry...SOS), a guanine nucleotide exchange factor that activates Ras. They expressed this OptoNB-SOS fusion protein...membrane-localized mCherry. With darkness, SOS binds Ras and initiates the extracellular signaling-regulated kinase (... -
How to Keep a Lab Notebook for Bioinformatic Analyses
TypeBlog Post...together. I also prefer the number system to using dates as labels, since I often work on multiple projects...sure that the original code remains in tact! Take notes Comment in your scripts: Comments on what a given...who are still learning the basics. However, these notes take up a lot of space and don’t tell you why you... -
3 Challenges in Plant Synthetic Biology
TypeBlog Post...infancy. A long timeline from transformation to testing and a lack of well-characterized genetic tools ...excited to see how this exchange of genetic tools facilitates plant science. Challenge #3: Intellectual property...in 2004, she accepted a commission in the United States Air Force. She left active duty to pursue a degree... -
Seven Tips for Using LinkedIn as a Scientist
TypeBlog Post...keep track of how you met the connection, as these notes are saved in the messaging stream. Unfortunately...LinkedIn is not the platform for politics, family updates, or click bait of any kind. For more on inappropriate...However, do comment on other people’s news and updates. Share your own good ideas, events and favorite... -
Troubleshooting Your Plasmid Cloning Experiment
TypeBlog Post...antibiotic in your plate might not work. Check the plates! Streak on them an E. coli strain known to be ...growing fine, it is probably time to prepare new plates with a fresh stock of antibiotic. Many small colonies... different temperature (30°C) for the growth on plates and in liquid culture. If you don’t get any positive... -
Hot Plasmids - March 2019 - Anti-CRISPR, 2in1 Cloning, Fluorescent Voltage Indicators, and Photoswitchable Proteins
TypeBlog Post...CASANOVA responds to blue light quickly. Within minutes of inactivating the CASANOVA complex, fluorescent...genes-of-interest (GOI) into two independent expression cassettes on a single plasmid (Grefen and Blatt, 2012). ...proteins that bind to either the light- and dark- states of the photoswitchable cPYP, which changes in conformation... -
The Breast Cancer Microenvironment: A Tumor’s Backstage Team
TypeBlog Post...caveolin 1, also known as CAV 1. Usually, caveolin 1 promotes the cell life cycle progression and enhances tumor... TAK 1, an enzyme that controls apoptosis and regulates transcription. Without TKA1, cells proliferate... 2004). In fact, Sanford H. Barsky and his team tested the influence myoepithelial cells had on the conversion... -
Choosing the B(right)est Fluorescent Protein: Photostability
TypeBlog Post... oxygen concentration may affect photobleaching rates (Shaner et al, 2008). This brings us to the key ...application (Goedhart et al, 2012). The third issue relates to the environment of the fluorescent protein. ...The issues with quantification of photobleaching rates can be summarized as follows: Instead of determining... -
CRISPRainbow and Genome Visualization
TypeBlog Post... cross-talk with the other orthologs. Ma et al. tested their dCas9 variants using gRNAs specific for telomeric...individual colors to 6. A gRNA with all three hairpins generates white light. All 6 gRNAs can be expressed in a...separate vector. Figure 2: CRISPRainbow generates 6 unique colors and a white composite. gRNAs with... -
Uncertainty about Labor Law Brings More Uncertainty to Postdoc Wages
TypeBlog Post...a new legal minimum of $47,476 per year, under updates to the Fair Labor Standards Act (FLSA). This was...22nd a preliminary injunction against the FLSA updates was granted nationwide. Again, we at Future of ...getting paid, which we will be analyzing for future updates. But suffice it to say, salaries are showing surprisingly... -
Summer SciComm: Preprints
TypeBlog Post...Photo courtesy of Henry Mühlpfordt, under a CC BY-SA license. Many scientists and advocates have argued...manuscript is drafted. Preprints were one of the key routes for researchers to share information about the ... -
Antibodies 101: Affinity Reagents
TypeBlog Post...large number of targets, and can require specific conjugates. And, of course, once you have them, you’ll need... along with a higher likelihood of aggregation (Bates, 2019). They are small enough to be produced in ...delivery systems. Elsevier Science & Technology Books. Bates A, Power CA. David vs. Goliath: The Structure, Function... -
Antibodies 101: Selecting the Right Antibody
TypeBlog Post...options. With limited time and budget you cannot test them all, so how do you know which ones to try? ...immune complexes are smaller than a primary:secondaries:conjugates complex. If you are trying to visualize.... The 5 pillars include genetic strategies that test expression in knock-out or knock-down down tissues...