...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...

...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...

...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...

...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...

...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...

...Updated Jun 1, 2021 by Meghan Rego. Reproducible data are key to science, so scientists are used to repeating...the DNA packaged inside the viral particles (Figure 1). Briefly, packaged DNA is extracted from purified...serotype. Additionally, we check viral transgene expression in transduced cells whenever possible. This post... for transgene expression. Once packaged in AAV, this pre-recombined DNA could lead to transgene expression... to these expected hits, we always get hits to “random” genes. Do these hits always mean the sample is...wanted to see how low we could go. We prepared a range of spiked samples from 0.1-20% and discovered that...find the optimal dose that allows for sufficient transgene expression but limits the likelihood of recombinase-independent...

...fluorophores are constantly being engineered. Table 1 lists a few common fluorescent proteins and their ...definitions found in Table 2 may not apply. Table 1: The specific mutations comprising common fluorophores...physiological processes. Re-engineering GFP to increase its range of color and application Almost as soon as its...backbones for constructing fluorescent fusions. Transcription Reporter: Placing GFP under the control of a...promoter in a given cell type. This type of transcription reporting was among the earliest uses of GFP...GFP. Fluorescence resonance energy transfer (FRET): This is used to study the interactions between two ...ion (such as Ca2+) concentrations and pH, using a range of strategies such as FRET, calmodulin, and others...

...In Biochimica et Biophysica Acta - Biomembranes (Vol. 1838, Issue 1 PARTA, pp. 15–33). https://doi.org...principles of fluorescent dyes/ligands Figure 1: Example structures of fluorophores, fluorescent dyes... dopamine D2/D3 receptors. Scientific Reports, 10(1). https://doi.org/10.1038/s41598-020-78827-9 Breen... of fluorescence: For applications that involve ranges of pH, and significant lengths of time, the stability...receptor conformation and signaling activity​​ (Sridharan et al., 2014). It's also crucial to evaluate how...the background. Dyes that can pass through cell membranes can prove useful for live-cell imaging. Experimental...considerations regarding the choice of fluorescent dyes (Sridharan et al., 2014). Manufacturers will typically recommend...

...for example, split cells on a Monday diluting them 1:10, you should be able to split on the following Thursday...Immunology. You can find Sana on LinkedIn.   References 1. Yin, R., et al. "Gene expression profiling analysis... are sensitive to pH. Anything between orange and light orange usually indicates that the cells have grown...culture media contain the pH indicator phenol red (pH range 6.0 (yellow) - 8.0 (red)). Blood pH normally lies...too young and should not be used for important transfection experiments right after they’ve been thawed.... spent organizing, an hour is earned (Benjamin Franklin)! It’s good practice to label your plates and/...

...CRISPR tools.  Distributing and depositing In Figure 1, you can see cumulative numbers of CRISPR plasmid ...new CRISPR depositors each year.    Figure 1: Addgene CRISPR distribution and deposits. (A) Cumulative...generate a classic double-stranded break, while modulators regulate transcription and chimeric efforts make...Countries shown are the top 10 requesting countries. FR=France; KR=South Korea; CH=Switzerland; AU=Australia.  ...Pyhtila et al., 2023). Our thanks to Rodolphe Barrangou for his authorial contributions to, and support...References Pyhtila, B., Kasowitz, S., Leeson, R., & Barrangou, R. (2023). The Expanding Dissemination and Distribution...

...listen to the interview with Tom McFadden!   1. Rosalind Franklin vs. Watson & Crick - Science History Rap...telling following the unfortunate story of Rosalind Franklin and her incredibly important role in solving the...

...cleavage (Harrington et al., 2017). Figure 1: Anti-CRISPR proteins (Acr's) can block CRISPR activity... ways to anti-CRISPR proteins in your experiments 1. Reduce off-target effects Prolonged Cas activity ... Knott GJ, Edraki A, Garcia B, Amrani N, Chen JS, Cofsky JC, Kranzusch PJ, Sontheimer EJ, Davidson AR,...695 . https://doi.org/10.3390/v10120695 Pawluk A, Amrani N, Zhang Y, Garcia B, Hidalgo-Reyes Y, Lee J, Edraki...

...editors (ABE) mediate A•T-to-G•C base changes (Figure 1), but it can be challenging to make these base changes...dCas to the genomic DNA of interest  Figure 1. When ABEs are in action they bind DNA so that the...the TadA domain can edit the short single-stranded DNA stretch that is accessible. Image from Gaudelli ... protospacer, the editing capacity of the ABE8s ranged from 1.5x higher for positions A5-A7 to 3.2x higher...References Gaudelli NM, Komor AC, Rees HA, Packer MS, Badran AH, Bryson DI, Liu DR (2017) Programmable base ...