...resolved loci with physical map distances of 75 and 2 Mbp, with the calculated fluorescent distances correlating...established physical map. In comparing pairs of targets ~2 Mbp apart, they noticed that they could evaluate the...to understanding how nuclear organization varies across the life of a cell, or how organization may be ...

...gene activation, with some genes experiencing around 2-fold activation levels. Although other methods have.... This method has been shown to activate genes at 2- to 3-fold higher than SAM, SunTag, and VPR (Zhou ...discovery of CRISPR/Cas systems, gene activation across multiple loci was an arduous process. When using...options to choose from when looking to activate genes across diverse cell lines.   dCas9-VP64 Description CRISPR... higher activation levels than SAM in some loci across Drosophila cell lines (Sajwan et al., 2019). SPH...

... of permeabilization and membrane integrity (Fig. 2). For example, saponin interacts with cholesterol ... at too high a concentration.      Figure 2: Examples of different permeabilization strategies...used cross-linker for IF. However, you might come across a couple other terms that can cause some confusion... just formaldehyde. Formalin: You may also come across formalin, which refers to a saturated solution ... cell membranes as they are not able to diffuse across natively. Permeabilization essentially punches ...

... colonies (150-200 per plate) across 20 fresh selective plates. Step 2: Express random phage DNA fragments... is also gaining a lot of new traction lately (1, 2), depends on knowledge of the structure(s) a given...microbiology 23.4 (2015): 185-191. PubMed PMID: 25708933. 2. Cooper, Callum J., Mohammadali Khan Mirzaei, and ... biology 34 (2015): 69-77. PubMed PMID: 26275959. 2. Weigele, Peter, and Elisabeth A. Raleigh. "Biosynthesis...

...resistance to the herbicide chlorsulfuron (Homo line 2) or wild type (WT), two weeks after herbicide was ... CLIPf tag and a C-terminal SNAPf tag; see Figure 2), the authors developed a cost-effective expression... when associated with microtubules. Figure 2: CLIP-SNAP fusion proteins for single-molecule FRET...authors used this platform to assay dozens of GPCRs across many cell types in varying conditions, revealing...

...(you generally don’t want more tag than protein), 2-3 tags is increasingly common. Tandem affinity purification...TEV cleavage site (more on that in a moment), and 2 ProtA IgG-binding domains. TAP has since come to encompass...found in this paper (available at Addgene).   Table 2: Protease recognition sites commonly used with tags...Guide Expression Vectors from the Berkeley QB3 MacroLab Eric thanks his wife, Annette Sievers, Ph.D.,...

...experiment using smFPs and standard neuronal tracers. 2. Visualization of neuronal sub-cellular structures...similar proteins. For example, N-cadherin (cadherin-2) is a postsynaptic cell adhesion protein that plays...PMID: 25915120. PubMed Central PMCID: PMC4573404. 2. Shepherd, G.M. (2004). In "The synaptic organization...Rizzo, 2009). With many different FPs that emit across the visible spectrum, researchers can also use ...

...pCRISPomyces-1 includes Cas9, a tracrRNA, and a CRISPR array, while pCRISPomyces-2 contains Cas9 and a gRNA ...and a subsequent curing protocol that requires only 2-3 hours incubation. E. coli and T. citrea scarless...gRNA cassette. The simpler system of pCRISPomyces-2 displays a higher editing efficiency, perhaps due to ...four major components including (1) a CRISPR RNA, (2) four proteins forming the QCascade DNA-targeting ...BbsI. This system results in 80-90% RNA knockdown across multiple gRNAs per gene (Singh et al. 2016). Sarah...

...from another within a fully stained sample. Figure 2 is an example of the emission spectra for two fluorophores...another during spectral unmixing.     Figure 2: The full emission spectra for BV421 and BV711 on ...marker. Given that the level of autofluorescence across various tissue types varies, it is critical to ... panel.     Figure 3: Similarity index across 12 different colors on a three laser spectral flow...

... a western, proteins are: (1) separated by size, (2) transferred to a membrane, and  (3) detected using...expensive system-specific sandwiches.   Figure 2: The western blot dry apparatus includes a stack of...vary the acrylamide content of the gel. For small proteins, use a higher percentage of acrylamide to increase... protein mix by size Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is the first...resolving gel composed of the crosslinked polymer acrylamide. In addition to the samples, a protein standard...Smaller proteins encounter less resistance in the acrylamide matrix and migrate faster through the gel. For... For large proteins, reduce the percentage of acrylamide. To further improve resolution, use a stacking...

...one injection step! The entire protocol takes about 2-3 weeks but requires less than eight hours worth of...for C. elegans genome modification. Figure 2: Schematic from Dickinson et al. (2015) illustrating...initial testing, Dickinson et al. tested this system across 7 genes in parallel; when they injected 60-80 animals...

...episode 2 "Organizing Literature" : I found out about Zotero here Dear Grad Student podcast episode 2 "Essential...LibreOffice, or Google Docs. Files can be synced across devices, and you can share your library with others...

...  AAV9, AAVrg pAAV-hSyn-DIO-mCherry 37825 AAV1, 2, 5, 8, 9 (20 µL size serotype testing) pAAV-CAG-GFP...size serotype testing) pAAV-CAG-GFP 50465 AAV1, 2, 5, rg (20 µL size serotype testing) pAAV-hSyn-EGFP...-ArchT-GFP (PV2527) 105679  AAV5 ppAAV-CKIIa-stGtACR1-FusionRed New tetracycline transactivators ...

...use of pAdEasy-2 (Addgene #16401) can increase the capacity of the rAdV vector. pAdEasy-2 does not contain... incorporate additional genomic deletions. Figure 2: First-generation rAdV vectors. Created with BioRender.com...and have to be handled at biosafety level two (BSL-2). They are highly immunogenic and can trigger a strong...era of personalized medicine . Genes & Diseases, 4 (2), 43–63. https://doi.org/10.1016/j.gendis.2017.04.001...adenoviruses using the AdEasy system . Nature Protocols, 2 (5), 1236–1247. https://doi.org/10.1038/nprot.2007.135...Adenoviral vector vaccine platforms in the SARS-CoV-2 pandemic . NPJ Vaccines, 6 (1), 97. https://doi.org...Adenoviral backbone plasmid that lacks E1 and E3. pAdEasy-2 Adenoviral backbone plasmid that lacks E1, E3, and...

...China, Korea, Japan, Brazil, India, and Mexico. Table 2: Addgene distributors Distributor Partnership...distribution after we partnered with them (Figure 2). In others, the distributor helped solve an issue...temperature for the duration of their journey. Figure 2: Annual distribution of items to countries with a ...materials both available and accessible to scientists across the globe?” By asking this question, and by listening...

...improve solubility and stability (Figure 2).      Figure 2: Crystal structures of CRBNmidi alone...fluorescent tags in Caenorhabditis elegans. Genetics, 228(2), iyae126. https://doi.org/10.1093/genetics/iyae126...mycobacteria, but such systems don’t always work across species due to differences in natural DNA repair...

...A-B+ cells. If you look at the dot plot in Figure 2, you might assume that all cell populations are positive... the different populations. In the case of Figure 2, one population is B- and another two are B+. Our ...are in the lower right corner.      Figure 2: This flow plot has two populations that are B+ and... the X axis may be presented. A horizontal line across the peak will indicate the cut-off value for a ...

...is unique compared to the rest of the genome and (2) the sequence is located just upstream of a Protospacer...pyogenes Cas9 variants with varying PAM sequences, 2) the use of Cas9 homologs derived from species other...Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System. Cell 163:759–771 . https://doi....When choosing a Cas9, remember to check that your tracrRNA and crRNA (or synthetic gRNA) are derived from...

...identifying the resources needed to accomplish that goal. 2. Identify the tools in that timeline Avoid writing...based on experimental timelines and specific aims. 2. Then make decisions based on financial values. All...Would I be able to get access to these materials?” 2. Always negotiate for a win-win decision An institute...early investigators making unnecessary short term sacrifices that hurt their goals, rather than focusing on...

...scientists have 1) the courage to make mistakes and 2) the integrity to recognize these mistakes quickly...She studies beta-cell function and failure in Type 2 Diabetes. Birmingham, AL, USA 33.5207°N 86.8025°W ...available to people who want to join the dialog and 2) remember that science should not be partisan: everyone...nightmare that is Times Square. But I made the sacrifice in April because devaluing science has alarming...reinforce their scientific interests. Our marches across the world have had an impact already, gaining additional...