...forming noncovalent dimers. This property of EGFP can lead to significant artifacts. If you're using green ...reportedly monomeric FPs are not actually monomeric, at least in practical terms. The propensity of a particular...FP dimerization coupled with POI dimerization can lead to polymerization of the fusion protein. A dimeric... members (superfolder, Emerald, CFP, YFP, BFP, Cerulean, Turquoise, Venus, and Citrine) can be monomerized...

...and controls (see Tips below), and by validating leads with other methods. More common are false negatives...levels of bait and prey proteins - overexpression can lead to false interactions. Lowered expression levels...techniques! Minimizing false negatives: Interactions leading to detectable reporter signals depend on a number...problems with one or more of these parameters can lead to a high number of false negatives in Y2H screens...

...a cooler. Chances are one of your colleagues has a frisbee at least. Use it! 8. Organize If you’re doing...make an effort to build relationships with their colleagues. It can be easy to get caught up in the minutia...true. If your event requires a lot of set up or cleanup, ask for volunteers to help out. Delegate- it will...

..., and they stepped up to apply for a grant which lead to their new career path.  A part of the career ...story for most established grant professionals is learning to succeed in grants through trial and error, ...increased their demand for stronger budgets with clear budget narratives, logic models, established partnerships... Throughout these moves and program changes, I learned to write grants for health, housing, economic development...

...research interests include proteomics, machine learning, and visualization for big biology. GPS for the...are critical? One hypothesis: the enhancers! This leads us to a three-step approach for designing an experiment...database. Aneesh's research interests span machine learning, proteomics, and user interfaces for math.     ...Catch Up on Your CRISPR Background with CRISPR 101 Learn about CRISPR Delivery Methods Validate Your Genome...

...numerous discoveries have been made using the Cas9 nuclease in the absence of genomic alterations. These studies...sequence via a targeting gRNA. But dCas9 does not cleave the DNA. Much of the research using dCas9 has focused...research conducted in the Hodaka Fujii Laboratory, please also see our interview with Dr. Fujii. References..., et al. "A programmable dual-RNA–guided DNA endonuclease in adaptive bacterial immunity." Science337.6096...

...innate immune system; and reconfiguring of the nuclear compartment, gene expression, and protein translation...responses that inactivate viral genomes or otherwise clear the virus from the cell population (Kafri et al....Adenovirus Tales: From the Cell Surface to the Nuclear Pore Complex. PLoS Pathog, 11(6), e1004821. https...stages of infection for efficient cell lysis and release of adenovirus from infected cells. J Virol, 70(...

...finding how-to videos on YouTube long before he learned (somewhat successfully) how to text or use Facebook... communication that you enjoy and that plays at least slightly to your strengths. Otherwise, you’re going...cost or freeware alternatives and how steep of a learning curve it will be to get to the necessary level...balance out cons. Flexible. Can allow people to learn in multiple ways from the same experience.  Can ...

... Make it clear that you are interested in the specific position you are applying to. Be clean and professional...letter, and if possible, send it to a mentor or colleague for a second look and helpful feedback!  What ...     Additional Resources on the Addgene Blog Learn to Develop Your Professional Network Become A Good...

...also reversible: once the environmental trigger leaves, gene expression is no longer activated. However...N2A, 3T3) and organisms (Chavez et al., 2016). To learn more about the different types of Cas9 activators....3149 Additional Resources on the Addgene Blog Learn how to Choose the Best Cas9 Variant for Your Next...More about Genome-wide Screens with CRISPR/Cas9 Learn How CRISPR Has Been Used with AAV Resources on ...

...and ssDNA cleavage by canonical base excision repair enzymes. At the same time Cas9n cleaves the opposing...editing of A•T to G•C in genomic DNA without DNA cleavage. Nature 551:464–471 . https://doi.org/10.1038/...and enrichment of DNA editing by APOBEC–Cas9 or cleavage by Cas9 in living cells. Nucleic Acids Research...

... Flow data is messy, so learning how to gate takes a lot longer than learning how to read a gating strategy...visualization tool, a quadrant will often be too clean for real-world data. Most plots will have a shape...    Of course, there are many more things to learn about when diving into flow cytometry data. There...

...A little over a decade back when Yamanaka and colleagues reported that it is possible to reprogram differentiated...fibroblasts and keratinocytes to peripheral blood mononuclear cells (PBMCs). PBMCs isolated from whole blood...Episomal vectors are based on the Epstein-Barr Nuclear Antigen-1 (oriP-EBNA-1) system where each vector... peripheral blood mononuclear cells...

...Cas13, the "collateral" activity of the enzyme can lead to off targets and result in significant, negative...processing pre-CRISPR RNA into mature CRISPR RNA and cleaving RNA at positions defined by the target:spacer ...limited to small deletions and small insertions, leaving a several kilobase-sized gap in the gene editing...Abudayyeh. Drag-and-drop genome insertion without DNA cleavage with CRISPR-directed integrases bioRxiv 2021. ...

...researchers recognize their MANY transferable skills learned while working in the lab and offering advice on...think of creativity as a form of innovation, it’s clear that creativity is absolutely a transferable skill...get distracted by an email or sidetracked by a colleague. To innovate, you’ll have to focus. Find yourself...(2019). Embrace your weird face your fears and unleash creativity. Gallery Books. Boyes, A. (2019, November...

... around, the plasmids are randomly positioned, leading to plasmid loss (Diaz et al., 2015).  Figure...designed small, high copy incompatible plasmids that lead to asymmetrical plasmid loss of the usually large...Addgene blog Read all of our Plasmids 101 blog posts Learn more about the origin of replication Find blog posts... Leah Schwiesow...

... proximity of the fluorophore and subsequently leading to a positive signal. The same effect can be achieved...this approach less sensitive in most applications. Learn About Fluorescent Biosensors Light-up aptamers (...assay. In 1999, Grate and Wilson paired laser cleavage and an RNA aptamer that bound the compound malachite...Addgene Blog Tips for Using FRET in Your Experiments Learn more about GFP Controlling Protein Activity with...

...elements, insulators, and silencers; however, some "leakiness" of transcription may occur. This is normally ...small RNA expression shRNA From the human U6 small nuclear promoter Constitutive  Murine U6 is also used, ...lacI or lacIq). Can be induced by IPTG or lactose. Leaky promoter with somewhat weak expression. lacIq mutation...the Addgene Blog Browse All Plasmids 101 Posts Learn about Mammalian Vectors Read about Reporter Gene...

...problems. This can lead to a lack of trust in the mentor and this training is clearly not equitable to ...assess the current state of research became more clear throughout the various sessions of the symposium...first couple of sessions of the symposium it was clear that, even though postdoc associations across different...

...degraded by uracil DNA glycosylase (UDG) and Exonuclease III (ExoIII) (step 4). UDG recognizes uracils...from newly synthesized DNA. Lack of both enzymes leads to a buildup of uracil and the insertion of uracil...limitation is sequencing depth and cost. You need at least 100-200 reads per variant, so the more codons you...to Generate Specific Mutations in Your Plasmids Learn How You Can Use CRISPR Pooled Libraries to Generate...