...three systems to analyze single cells from a mixture (3): An optics system A fluidics system An electronics...expression. A routine flow cytometry experiment uses 2 or 3 different fluorescent colors each measuring a different...biosafety cabinet with cloning rings or following labor-intensive limiting dilution protocols? If this sounds...fluorescent microscopy must be used. To learn more about the most suitable fluorescent microscopy technique...a laser and the instrument collects information about the cell’s size, complexity and fluorescence intensity...captured by detectors and provides useful information about the cell. Light that continues in the same direction... forward scatter (FSC) and provides information about the relative size of the cell; larger cells produce...

...preparation, and lead your own research project.  3. Do you prefer being a generalist? Working on a variety... Resources Resources on the Addgene blog Learn about career planning resources What Do I Do Now? Academic...

...Palindromic Repeats, in Pubmed returned 728 articles (3/12/2014). With so many options for CRISPR plasmid ... gRNA design tools Need more questions answered about CRISPRs? Check out the full list of 16 FAQs answered...

...identifier for individual cells. Each cell is labeled with 3-4 tags to increase the diversity of the CellTag library...Additional resources on the Addgene blog Learn more about induced pluripotent stems cells (iPSCS) and reprogramming...

...Reference Rq(R165L) Human M3 muscarinic Arrestin-2/-3 CNO* Increase Arrestin translocation Arrestin signalling...activity. PSAM-5HT3 LGICs pair a PSAM domain with a 5-HT 3 serotonin receptor sodium- or potassium-selective ...K + ions, and activates neuronal activity. Figure 3: PSAM4-based ligand-gated ion channels, their effect...routes. Image courtesy of Ute Hochgeschwender . Table 3. Descriptions of LMOs and their activity in neurons.... However, CNO is back-metabolized to clozapine and other clozapine metabolites and these can have off-target...concentrations of clozapine N ‐oxide (CNO). CNO is a metabolite of the antipsychotic clozapine and seems to be...experiments in this species may be entirely due to back-metabolism of CNO to clozapine, demonstrating the need for...

...tagged with different fluorescent proteins or dyes. 3. Two-photon microscopy Two-photon excitation microscopy...PMID: 18718768. PubMed Central PMCID: PMC2569002. 3. Egen, Jackson G., et al. "Macrophage and T cell dynamics...fluorescence microscope where the light source is mounted above (epi) the specimen and the excitation light passes...technique that allows imaging of living tissue up to about one millimeter in depth. Two low-energy photons ...response, but the study of these dynamics was still laborious as epifluorescence microscopy wasn’t able to give...skin. Two-photon intravital microscopy. The video above depicts the infiltration of neutrophils expressing...

...(i.e. optical sectioning (2) or super-resolution (3). Fluorescence microscopy techniques   Fluorescent...methods 2, 920-931 (2005). PubMed PMID: 16299477. 3. Eggeling C, Willig KI, Sahl SJ, Hell SW. Lens-based...call Epi-fluorescence microscopes. (See left image above for an example image). Point Scanning Confocal Microscopy... plane to reach the detector and all light from above or below is excluded. Parallelized Confocal Microscopy...due to limitations in light penetration mentioned above (5). Thick static samples Ex: Fixed tissue sections...sectioning. Point scanning confocal microscopes (see above) often provide the highest quality images for this...

...-mediated marker excision, it takes approximately 3-4 weeks to obtain a homozygous, outcrossed knock-in...produced a higher editing efficiency, while for edits 3’ to the PAM sequences corresponding to the opposite...learn more about genome editing with CRISPRs? Check-out Addgene's CRISPR Resources Learn about gRNA design...interest, I encountered low efficiency (0.13%) and laborious handling (screening 768 F1 animals). Recovering...

...strand completely; it is essential, however, that the 3’ end of the primer corresponds completely to the template...proceed. Usually a guanine or cytosine is used at the 3’ end, and the 5’ end of the primer usually has stretches...stretches of several nucleotides. Also, both of the 3’ ends of the hybridized primers must point toward ...restriction site at the 5’ end of your primer, note that a 3-6 base pair "clamp" should be added upstream in order...amplification process. When designing, if unsure about what nucleotide to put at a certain position within...capabilities. Taking into consideration the information above, primers should generally have the following properties...

...assist with restriction enzyme digestion (usually 3-6bp). Restriction Site: Your chosen restriction site...Primer will use the sequence 5'-ATGTGGCATATCTCGAAGTAC-3' for the region that binds the ORF and we will add...our Forward Primer 5'-GAATTCATGTGGCATATCTCGAAGTAC-3'. Many restriction enzymes do not cut DNA efficiently...more information). Thus, we recommend that you add 3-6 bases upstream of your restriction site to improve... sequence of 5'-TAAGCAGAATTCATGTGGCATATCTCGAAGTAC-3'. For the Reverse Primer, the design is similar, but...including the stop codon (5'-TGGCATATCTCGAAGTACTGA-3'), then adding NotI (GCGGCCGC) and then TAAGCA to ...sequence of 5'-TGGCATATCTCGAAGTACTGAGCGGCCGCTAAGCA-3' (30bp with 18bp of homology to the ORF). We now ...