...yes, activate S. pyogenes Marraffini pSimpleII-U6-tracr-U6-BsmBI-NLS-NmCas9-HA-NLS(s) 47868 Mammalian U6...

...again but store at 4 °C and use within 1 week. Keep track of the Ct value for each standard over time. They...

...Anti-Cas9 Western blotting was performed on whole-cell extracts from the initial stable cell pool (pool) and from...

... milk going to help me extract DNA from agarose gel?!”   The DNA gel extraction with glass milk method... a similar workflow as a column-based plasmid extraction kit. First, you lyse the bacteria and denature...contamination and you can use a phenol chloroform extraction to remove remaining proteins and RNase. Finally...ready for use! Plasmid purification and DNA gel extraction with glass syringe filters Starting material:...fragments The plasmid purification and DNA gel extraction method described in Kim and Morrison is nearly...protocol and guanidine isothiocyanate for the gel extraction protocol) disrupts the DNA’s hydrogen bonds with... more flexible than commercial kits.  DNA gel extraction with glass milk Starting material: agarose gel...

...pre-amplified stock. A last option is to perform a gel extraction of either the original sample or the amplified...

...information from a region (anterograde tracer, Fig. 1B). Tracers are typically delivered to a particular... either trace downstream from the soma (neuron cell body) toward the axon (anterograde tracer) or upstream...making them useful tracers, but finding a virus that works well for retrograde tracing can be difficult....brain works. There are two methods of neuronal tracing (Fig. 1): one to visualize neurons that send information...information to a region (retrograde tracer, Fig. 1A), and another to visualize neurons that receive information...from the axon terminals to the soma (retrograde tracer) (Fig. 2). Viruses as neuronal scalpels Viruses...transport and can therefore perform retrograde tracing, but often at the cost of toxicity, low expression...

...days. The crRNA and tracrRNA are annealed on a PCR block and the annealed cr:tracrRNA is then mixed with...Anneal crRNA and tracrRNA using a PCR block. Add Cas9 protein to the annealed cr:tracrRNA and incubate at...Tag 96 genes at a time using Custom Synthetic cr:tracrRNA/Cas9 Ribonucleoprotein (csRNP) delivery Epitope...started off by using custom synthetic two-part cr/tracrRNAs for CRISPR experiments (in collaboration with ...-tagging experiments. Cas9 protein, annealed cr:tracrRNA, and single-stranded donor DNA. The success of...have recently demonstrated that the two-part cr:tracrRNA system is much more potent than the in vitro-...Dewari et al. 2018 and Figure 2). Not only is the cr:tracrRNA approach highly efficient, it is also straightforward...

... challenging to track intracellularly, but aptamers permit the study of intracellular RNA dynamics. Aptamers...its ability to track RNA dynamics in a model of Fragile-X tremor/ataxia syndrome (Strack et al., 2013)....target cellular RNA to specific intracellular locations using Cas9 and track the localization of the complex... molecule. Fluorescent RNA aptamers are used to track cellular processes by transforming cells with plasmids...then, Spinach2 has been used to investigate intracellular viral DNA dynamics in neuronal cells (Nilaratanakul...is better able to maintain its fluorescence intracellularly due to a lower dependency on magnesium and ...bright, high-affinity complex for RNA labeling and tracking." ACS chemical biology 9.10 (2014): 2412-2420....

...video abstracts identified. The journal Cell had the largest average view count per video abstract at 2,947...antibiotic prescriptions for respiratory tract infections. The video abstracts provide a brief introduction to ...science communication through video     Video abstracts about research, especially published research,... views) rounded out the top four average video abstract view counts.” For researchers looking to garner...garner more attention for their work, a video abstract may be a useful tool for turning video views into ...there was a growing number of online research abstracts. With journals in medicine, physics, math, and...), I created a new platform for sharing video abstracts about research: www.WeShareScience.org. WeShareScience...

...of common genetically encoded neuronal tracers Neuronal tracers such as epitope tags and fluorescent proteins...molecules in your favorite neuronal population and track connections to that population. Limitations of ... used for protein localization, isolation, and tracking experiments (Rizzo, 2009). With many different...Spaghetti Monster - A new and improved neuronal tracer With the limitations of epitope tags and fluorescent...fluorescent proteins, it has been crucial to develop new tracers that enable accurate labeling of neurons. As discussed...expressed proteins in particular are difficult to track with these classic probes. To overcome the limitations...Addgene Uses of smFPs 1. Multi-channel connectomic tracers Fluorescent proteins such as GFP have been used...

...wanted poster that Cas9 uses to track down its suspect—the target DNA. tracrRNA: A “trans-activating CRISPR... of CRISPR. The bacterial chromosome encodes a tracrRNA (in some systems including Cas9), Cas proteins...here). The repeat sequence base pairs with the tracrRNA, and the spacer sequence is used to target complementary...laboratory settings, an sgRNA includes the crRNA and tracrRNA sequences in a “single-guide RNA” that performs... crRNA, forming the repeat:antirepeat duplex. tracrRNAs also contain multiple stem loops that are recognized...proteins, like LbCas12a or AsCas12a, do not require a tracrRNA. sgRNA: “Single-guide RNAs” were invented by synthetic...synthetic biologists. An sgRNA combines a crRNA and a tracrRNA into a single strand that folds back on itself...

.... Developmental biology insights using lineage tracing in zebrafish To highlight some of the features ...zebrafish useful, I’ll outline two recent lineage tracing approaches that led to important insights in developmental...developmental biology. Microscopy-based lineage tracing Over a period of 24 hours, the externally fertilized...cells in the developing zebrafish embryo have been tracked using microscopy and advanced computational approaches...Keller et al., 2008). This microscopy-based lineage tracing defined a high-resolution map of early embryogenesis... mutagenesis screens. Microscopy-based lineage tracing can help illuminate how signaling pathways—such...development. Single cell sequencing-based lineage tracing Recently, gene expression in all cells of the developing...

...available for tracking certain cell types. Another useful tool for marking and tracking cells is the the...fluorescently labelled antibodies made it possible to track cells from one organ to another. Thus several labs...lymphoid organs did in the past. How do researchers track fluorescently labeled cells? 1. Epifluorescence ...fluorescent proteins gave researchers the ability to track immune cells within organs and visualize how cells... to the immune lineage, scientists could easily track GFP expressing cells. For example, by harvesting...after antigen challenge, scientists were able to track where GFP labelled B cells were in mounted slices...organs. These GFP labelled cells could also be tracked by cell sorter giving immunologists the ability...

...its native context, the tetracycline repressor (TetR) can bind to the tetracycline operator sequences (TetO...preventing transcription. In the presence of tetracycline (Tet), TetR preferentially binds Tet over the...repressible system, Gossen and Bujard created the tetracycline-controlled transactivator (tTA). They fused ...TRE)), allowing transcription to proceed. When tetracycline or one of its derivatives is added, it binds...information on Addgene Tet plasmids, see our Tetracycline resource page. Cumate Switch Massie et. al developed...permit exquisite control of gene expression and tracing of gene expression across development. One common...control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc Natl Acad Sci U S...

...a look at some popular self-labeling tools: Tetracysteine: The first reported chemical labeling tag, from...Commercial ligands available Ease of use Tetracysteine (Cys4) 15 Biarsenical dyes (FlAsH, ReAsH... HaloTag for their “ticker tape” method for intracellular recordings, switching between different colored...time points.   Figure 3: Single-molecule tracking of actin filament assembly-regulating proteins...appearance at the filament tip from (a). c) Intensity traces at the tip of the filament from (a-b), showing ...length (lower). d) Intensity and filament length traces for a different event where CP appears briefly ...labeling specificity than short peptide tags like tetracysteine (Jing & Cornish, 2011). HaloTag and SNAP-tag...

...difference. Traces The Traces tab shows the edited and control, non-edited Sanger traces in the region...as determined by comparing the edited trace to the control trace. In the ICE algorithm, potential editing...and Sanger sequenced. ICE compares these sequence traces to give a detailed analysis of CRISPR editing. ...three tabs - Contributions, Indel Distribution and Traces - provides particular details about the indel profile...the reference sequence derived from the control trace file. On the plot, the green (edited sample) and...calls from the .ab1 file are also shown above each trace. The horizontal black underlined region represents...

... epitopes of interest that are found in the extracellular space. The targeted epitopes can be motifs within... T cell. Created using biorender.com.   Intracellular labeling Oftentimes, a marker of interest is... cannot penetrate the cell membrane; hence, intracellular labeling with antibodies requires chemically...analysis to be shifted to a later day. Note that intracellular labeling must be performed only after viability...staining and surface labeling are already done. Intracellular labeling can be subdivided into two categories...procedure in that case.     Figure 3: For intracellular labeling cells need to be fixed and permeabilized...cell proliferation and cell viability. Unlike intracellular labeling, dye staining can be done on live cells...

... beliefs. I asked participants to read short abstracts comparing the mortality rates between two controversial...peer-reviewed study. The second group read the same abstract but with the names reversed, meaning that the ...reversed. The third group read a version of the abstract where the real medical conditions had been swapped...asked to evaluate the quality of the study those abstracts represented. The point of this study was to see...see if their evaluations of the abstracts were driven by the quality of the study design or by their preconceived... significantly more favourable ratings to the abstracts that affirmed their personal beliefs even though...identical. Likewise, when participants read an abstract for a study with findings that challenged their...

... and recent advances have allowed scientists to track multiple genomic loci over time using all the colors...CRISPRainbow. Figure 1: CRISPRainbow was used to track the localization of 6 chromosome-specific loci in...labeled dCas9 to a given genomic locus, you can track the localization and movement of this locus in live...depositor Stanley Qi and used in Chen et al. to track telomere dynamics throughout the cell cycle. To ...distances from 2-75 Mbp. Labeling multiple, intrachromosomal loci Building on the work of Chen et al., ...turned their attention to mapping pairs of intrachromosomal loci. The technique successfully resolved ... this work represents the first mapping of intrachromosomal loci, a major benchmark in characterizing ...

...but it is for a scaleable lab. When you need to track the location, quality, growth, and legal status ... that everything is grown successfully, we must track and manage all of these steps. Unfortunately, biology...they needed to, they’d have to individually keep track of any failures and do troubleshooting on their ... it’s monumentally difficult without software. Tracking lab procedures also gives you access to hard data...repeated. We also now have a good interface to begin tracking analytics data for lab procedures. With this data...are distinct from their contents. You should be tracking both, you should easily be able to move contents...lab team about how they would like these things tracked. Terminology and language are tricky Names for ...