... 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 ...

...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...

...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...

...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 ...

...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...

...of ~75% in response to the magnetic field (Figure 2); they named this variant MagLOV. Andrew was thrilled...didn’t need any added cofactors.      Figure 2: Fluorescence intensity and ΔF/F of MagLOV, a variant...sample. The imaged area measures approximately 82 mm across. Video courtesy of Andrew York and Maria Ingaramo...new microscopy tools for biological applications across a wide range of topics. He embraces open science...

...antibiotic selection. Essentially, this gene is a 2-in-1 providing both bacterial and eukaryotic selection...Zeocin-induced DNA cleavage in human cells. Mutagenesis, 20(2), 111–114. https://doi.org/10.1093/mutage/gei016I ...because it can be a one-stop shop for selection across systems and species. The cost? Zeocin is genotoxic...off-target mutations are undesirable. Pros: Works across many species/systems. Cons: Doesn’t work for all...

...muscular dystrophies, heart disease, and type 2 diabetes, across four different types of human cells, including...(#186715) and hCtRNA_VT (#186716).   Fig. 2: Comparison of previous representative dual-gRNA architectures...

...is always best to have options.      Fig. 2: Addgene’s western blot for mEGFP-HRas (from Plasmid...the presence of oxidizing agents. Adapted from Fig. 2 Campbell et al, Nature Methods   Find YFP plasmids...cytoplasmic mRNA with 90-99% knockdown efficiency across the 11 candidate transcript targets analyzed. ...