...  Hot plasmids covered in this segment Hot Plasmid 1: Photocleaveable PhoCl protein for controlling molecular...Hot Plasmid 3: CRISPR base editors with enhanced range and specificity. Listen to to the Segmennt of Improved...

...Hot Plasmid 1: The Tol2 Gateway-Compatible Toolbox for quickly generating zebrafish transgenic lines to ...

...874 . https://doi.org/10.1038/nmeth.3993  Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu.../doi.org/10.1038/s41586-019-0908-x  Nishimasu H, Ran FA, Hsu PD, Konermann S, Shehata SI, Dohmae N, Ishitani...949. https://doi.org/10.1016/j.cell.2014.02.001  Ran FA, Cong L, Yan WX, Scott DA, Gootenberg JS, Kriz...indel formation and phenotypic changes as early as 1 week post-injection. Livers from these mice were histologically...gene delivery due to their low immunogenicity and range of serotypes allowing preferential infection of ...prime editors. While using AAV to deliver large transgenes might seem daunting, it’s actually a challenge...split AAVs. In general split AAVs break a large transgene in two pieces and package each piece into an individual...

...all of its successes, has many deep-rooted flaws (1). As we discovered through the four workshops at the... Follow David on Twitter @driglar.     References 1. Alberts, Bruce, et al. "Rescuing US biomedical research...Ambika, et al. "Transforming training to reflect the workforce." Science Translational Medicine (2015)...better solution would be to reduce the number of entrants into PhD programs to more closely reflect the ...symposium. Eve Marder, professor of Biology at Brandeis University, has recently published an article ...Among other involvements, he helped to establish grants through the Sloan foundation for postdoc-related...

...2021.   The AID system relies on a transport inhibitor response 1 (TIR1) expressed under a desired promoter...and accurately in a “single pot.’   Figure 1: Single-Pot Assembly: a) Graphic depiction of chromatin...editing assemblies are sensor-actuators, either transcriptional regulators or chromatin-modifying enzymes. ...cell surface. When the cells interact, GFP is transferred to the receiver cell, and the receiver cell turns...These libraries encode target sites flanked by randomized nucleotides in place of a protospacer adjacent.... Check out the new additions.  Image calcium transients in PV interneurons with AAV-S5E2-GCaMP6f, now...

...scavenger hunt and the subsequent clue. (Begin with clue 1 below!) You will find five photos this way. At the...Subscribe to the blog to stay in the know!     No pranks, just fun and games this April Fool’s Day on the... to find pictures of Addgene pets with Addgene-branded clothing on the blog. Below each photo, the pet...

...company culture.  It’s also a good idea to prepare 1-2 unique questions for each interviewer by looking...to take notes after each interview.   Tailor your 1-hour presentation to your audience Start by asking...position, I just had a phone interview. I also transitioned between different lab groups without having ... an opportunity to demonstrate technical and transferable skills that make you qualified for the position...experience less stressful. First, you can make arrangements with those you live with to have a quiet place...

...screening blog post for more information.   Figure 1: Result of a blue-white screen. Image from Stefan ...Because Sanger sequencing can usually sequence just 1 kb of DNA, custom primers are especially helpful in...information (more on this below). Plating the transformation on ampicillin plates containing X-gal allows...

...plasmid, based on the assumptions outlined in Table 1.   Of course, this is for the simple insertion of...any restriction enzymes or vector purchases (Table 1). Restriction enzymes and vector purchases can raise...relevant work in your field! However, there’s no guarantee you’ll get a response on the first try (or any...

...healthcare system was $35.2 billion (or $140 per $1 invested in the trial). The goal of gendered innovations...is the goal of Gendered Innovations. This project: 1) develops state-of-the-art methods of sex and gender...results, but this result shouldn’t be taken for granted for all research going forward. It’s just not that...

...all the colors of the CRISPRainbow. Figure 1: CRISPRainbow was used to track the localization of...is available as a kit from Addgene.   References 1. Chen, Baohui, et al. “Dynamic Imaging of Genomic ...spatial relationship of the two genes over time, at a range of distances from 2-75 Mbp. Labeling multiple, intrachromosomal...a system has been previously described for transcriptional activation/repression and genome editing. ...

...on DNA methyltransferase 1 (DNMT1) to copy methylation marks from parental to daughter strands of DNA....modifications). Ready to get started?   Figure 1: Break free from the ball and chain!   Epigenetics...Tethering dCas9 to the histone acetyltransferase p300 or the DNA methyltransferase DNMT3A allows researchers...affect transcription independent of their enzymatic activity. They interact with transcriptional machinery...specifically edit methylation sites.  Step aside, transcriptionally active dioxygenase  When I started to work...by distinct proteins and result in unclear transcriptional consequences (Sprujit 2013). Enzymatic domains...in the genome and directly interacting with transcription machinery.   Well, I’ve got some good news...

...for the transcriptional activation of endogenous genes and it consists of three components: 1. A nucleolytically...complex with guide RNA and target DNA. Nishimasu H, Ran FA, Hsu PD, Konermann S, Shehata SI, Dohmae N, Ishitani... sgRNAs for each transcription start site in 15,977 human genes. The transcriptional repressor (CRISPRi...The next insight was to assemble a synthetic transcription activation complex consisting of multiple distinct...together synergistically, modeled after natural transcription activation processes. Konermann and her colleagues...consequences of repressing or inducing individual transcripts. The activator (CRISPRa) sgRNA library uses the...CRISPRi) library contains 10 sgRNAs for each transcription start site in those 15,977 human genes. Weissman...

...why he chose to go into genome engineering. Chapter 1: Early research on CRISPR and what it's like to work...competitive field. Chapter 2: Wei Leong Chew's swift transition from graduate work to an independent position... Coming up with new project ideas, applying for grants, and mentoring. Chapter 5: Wei Leong Chew's lab...

...the lab running like a well-oiled machine. Lab tip #1: Glove trick Putting on lab gloves can sometimes be...hopefully make your life a little easier. These can range from technical tips as in our episode on Barcoding...

...overhangs may be present in the original plasmid (Option 1) or added using PCR-based amplification (Option 2)...Methods Mol Biol. 2014;1116:119-31. doi: 10.1007/978-1-62703-764-8_9. PubMed. More Resources at Addgene:  ...vector + insert may spontaneously religate, this transient construct retains functional Type IIS sites and...

...depths (1 mm), important for certain applications such as in vivo tissue imaging.   Figure 1: Ace-mScarlet.... They can be targeted to the plasma membrane to measure membrane potential and can also be directed to... the membrane), the voltage sensing component (i.e., rhodopsin naturally resides in the membrane), or ... of voltage gated ion channels all depend on transmitting and sensing electrical potential. Thankfully... want to monitor the voltage change across the membrane of cardiac cells. Similarly, neuronal signaling...propagated through multiple cells to do things like translate perceived signals to physical actions. Understanding...rhodopsin proteins were initially evolved for ion transport, not fluorescence, they are not nearly as bright...

...interrogated by the optical system (Figure 1).   Figure 1: Cells expressing a particular surface ...populations of cells in a complex tissue or sample (1). When combined with cell sorters, this technology...permeabilize the cell membrane to allow antibodies to pass through the cell membrane. Formaldehyde fixation...dye emits green, yellow and orange photons while PE emits yellow and orange photons. If FITC and PE were...sources that allow users to assess a cell’s size, granularity, and target protein composition. It is used for...target protein expression levels, assessing post-translational modifications, determining cell health, analyzing...a pressurized buffer called sheath fluid and transported through tubes or capillaries to a laser. As the...

...and placed in a buffer. Antibody Structure Figure 1: Structure of an antibody A standard antibody is made... together to form a “Y” shape, as shown in Figure 1. The two arms of the Y structure are responsible for...proteins by size. Transfer proteins from the gel to a membrane. Incubate the membrane with an unconjugated...block the membrane to remove excess antibody and prevent unwanted binding. Incubate the membrane with a ...Wash and block the membrane. Activate the HRP with a substrate kit. Image the membrane using either X-ray...hybridoma cells into plasmids. Plasmids are then transfected into cells and induced to express antibodies....variable regions, shown in light blue and light orange, that bind to a specific antigen, triggering an...

...detects samples at clinically relevant concentrations (1-3 fM; 1x106-fold lower than before), and our combined... health, energy, and sustainability.   References 1. Pardee, Keith, et al. "Rapid, Low-Cost Detection ...fast-tracked development of diagnostics. Mostly transmitted by Aedes mosquitoes (aegypti and albopictus) ...and electronic companion technologies that has transformed the assay’s capabilities. Specifically this includes...