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We narrowed to 492 results for: ara-2

Showing: 101 - 120 of 492 results

  1. Grad School Advice Part 1: Picking a Lab and a Project

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    Blog Post
    ...Introduction from Tyler 1:13 - 2:02 : Niroshi and Ben introduce themselves 2:03 - 3:22 : How did you end ...things get tough in the dreaded third year. Find Part 2 Here! We'd like to thank Niroshi and Ben for taking...senior graduate students, Ben Vincent and Niroshi Senaratne, from the Biological and Biomedical Sciences program...Biology, and he tweets @benjvincent.     Niroshi Senaratne is a graduate of the PhD program in Genetics and...

  2. CRISPR 101: Targeting RNA with Cas13a (C2c2)

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    Blog Post
    ...question: C2c1, C2c2 and C2c3 (short for Class 2, candidate 1, 2, or 3). C2c1 and C2c3 are related to Cpf1,...et al. "Discovery and functional characterization of diverse class 2 CRISPR-Cas systems." Molecular cell...however cutting efficiency of Cas13a is reduced when 2 mismatches are present. The protospacer flanking sequence...PMC5127784. Find plasmids from this paper at Addgene. 2. East-Seletsky, Alexandra, et al. "Two distinct RNase... Leptotrichia shahii (LshCas13a) to begin to characterize this new effector (Abudayyeh et al., 2016). ... plant cells (Abudayyeh et al., 2017). They characterized Cas13a from Leptotrichia wadei (LwaCas13a), ...multiplexing; expressing 5 gRNAs simultaneously produces comparable gene knockdown to single gRNA expression. Although...

  3. Viral Vectors 101: Optogenetic Tools

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    Blog Post
    ...derived from direct comparative analysis of microbial opsins. Nature Methods, 9(2), 159–172. https://doi.org...for controlling neuronal activity, channelrhodopsin-2 (ChR2), a blue light-activated cation channel, from...used in tandem with the opsin.      Figure 2: Diagram of excitatory (ChR2) activation by light ...) domains, phytochrome B (PhyB), and cryptochrome 2 (CRY2) (Zhang & Cui, 2015). When stimulated with light...Hegemann, P., & Bamberg, E. (2003). Channelrhodopsin-2, a directly light-gated cation-selective membrane ...intracellular signaling pathways. Trends in Biotechnology, 33(2), 92–100. https://doi.org/10.1016/j.tibtech.2014.11.007...

  4. Plasmids 101: Dimers and Multimers

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    Blog Post
    ...identification.   Fig. 2: Gel image of Plasmid 61564: pBAMD1-2 (~4.7 kb) digested with SpeI and... at ~3.6 kb and ~1.1 kb (lane 3). Uncut DNA (lane 2) has a band at <3 kb, which corresponds to the supercoiled...formation of plasmid multimers. Cell. 1976 Dec;9(4 PT 2):707-16. doi: 10.1016/0092-8674(76)90134-3. PMID: ...dimers in Escherichia coli K-12. Plasmid. 1989 Mar;21(2):138-41. doi: 10.1016/0147-619x(89)90057-7. PMID: ...Plasmids and Chromosomes. Microbiol Spectr. 2014 Oct;2(5). doi: 10.1128/microbiolspec.PLAS-0025-2014. PMID...Behavior of Plasmid DNA. Microbiol Spectr. 2015 Apr;3(2):10.1128/microbiolspec.PLAS-0036-2014. doi: 10.1128...: 21285356; PMCID: PMC3060453. Bazaral M, Helinski DR. Characterization of multiple circular DNA forms...

  5. Tips for a 1st Time CRISPR User (by a 1st Time CRISPR User)

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    Blog Post
    ...transductions and use a range of reasonable MOIs. 2. Create a monoclonal Cas9-expression cell line. Especially...doing a screen. I made a few monoclonal lines (Figure 2) and I was surprised at how variable Cas9-expression...across the different cells (Figure 3).   Figure 2: Cas9 expression in monoclonal cell lines generated... expression Overview: By Western blotting (Figure 2, pool), I knew the cells expressed Cas9, but I didn... appear green despite having active Cas9 (see tip 2). Pro-Tips 1. To quantify Cas9 activity using this...Western blotting against GFP expression, but I didn’t. 2. In the original publication they see GFP expression...and I got clear bands using the conditions above. 2. Design your primers so that the digestion site isn...

  6. Flow Cytometry Readouts: Yes, No, and Everything in Between

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    Blog Post
    ...low expression, and everything in between (Figure 2). These smears tend to occur when you are looking ...based on your experiment. You can see from Figure 2 that determining where to place your gate based on...the dot plot would be complicated.  Figure 2: (A) a smeary dot plot showing a range of antibody...plot in Figure 4 compared to the dot plot in Figure 2. Quantification using MFI makes it easier to identify...positive samples are clearly distinguishable as two separate populations. Created with biorender.com. Pro...with the baseline suggesting this sample has a comparable expression to the baseline/negative control. ...expression, using a histogram plot and MFI values to characterize your samples may allow for better interpretation...

  7. Genome-wide Screening Using CRISPR

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    Blog Post
    ...gRNA-containing plasmid, or a 2-plasmid system in which Cas9 must be delivered separately.  Mutant cells are enriched...targeting a specific gene within the genome (see Fig. 2). Individual gRNAs are designed in silico using publicly...mouse, human, fly, E. coli, and T. gondii genes.  2) What genetic modification are you trying to make?...CRISPR libraries is a multi-step process (see Fig. 2). In most cases, CRISPR libraries are provided at ...uncontrolled cell growth, such as cancer. Figure 2: The CRISPR library must be amplified (A) to be used...If you've obtained a ready-to-use lentiviral preparation from Addgene, you can skip the steps above! Cells...

  8. 22 Hot Plasmid Technologies from 2014

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    Blog Post
    ...Currently available from the Kim lab are 2 presynaptic and 2 postsynaptic targeting mGRASP plasmids. Additionally... cells were pulsed with 2µg/ml of Doxycycline for 2 hours, and imaged on the Keyence BZ-9000 fluorescence...work! Robinson et al., Dev Cell 2010 Feb 16;18(2):324-31. Cheeseman et al., J Cell Sci 2013 May 1...promotes the formation of an isopeptide bond between 2 peptide tags, SpyTag and KTag. The group demonstrated... of three sets of cloning vectors (Level 0, 1, or 2) which can be utilized in three successive assembly...1 modules can be similarly assembled into a Level 2 vector, thus forming a functional genetic circuit....circuit. Flexibility has been built into the Level 2 vectors to allow for additional iterations of Level 1 assembly...

  9. Using AAV for Neuronal Tracing

    Type
    Blog Post
    ...Baekelandt, V. (2007). Comparative analysis of adeno-associated viral vector serotypes 1, 2, 5, 7 and 8 in mouse...Yamamori, T. (2015). Comparative analyses of adeno-associated viral vector serotypes 1, 2, 5, 8 and 9 in marmoset...for retrograde transport has been found for AAV1, -2, -5, -7, -8 (Taymans et al., 2007), AAV1 (Hollis et... tropism and axonal transport of AAV serotypes 1, 2, 5, 6, 8 and 9 in the mouse brain. PLoS One 8:e76310...reveal novel hippocampal CA2 circuits. Nat Neurosci 17(2), 269–279. PubMed PMID: 24336151. PubMed Central PMCID... Mechanisms of Motility and Regulation. Neuron 84(2), 292–309. PubMed PMID: 25374356. PubMed Central PMCID...transduction efficiency of recombinant AAV serotypes 1, 2, 5 and 8 in the rat nigrostriatal system. J Neurochem...

  10. Fluorescent Proteins 101: Visualizing Subcellular Structures & Organelles

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    Blog Post
    ... to the lysosome where they are finally degraded (2, 3). Once you've created your fusion protein, it's...examples include the ImageJ plugins JACoP and Coloc 2, BioImageXD, and customized CellProfiler pipelines...colocalization analysis example is given in Figure 2 (adapted from 4) - Dunn et al. 2011 provides an in-depth...Since colocalization relies upon the detection of 2 independent fluorescent signals and their potential...here! References 1. Allen Institute Plasmid Pages 2. Cooper GM. 2000. The Cell: A Molecular Approach. ...Therefore, it can be beneficial to employ well-characterized marker proteins tagged with FPs to highlight...spectra of the selected FPs need to be sufficiently separated, most commonly FPs with red and green wavelengths...

  11. New Neuroscience Tool: The iGluSnFR3 Glutamate Sensor

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    Blog Post
    ...kinetics which are illustrated in figures 2 and 3.  Figure 2: Glutamate titration curves for the different...signals upon sensing glutamate using both 1-photon and 2-photon imaging.   Figure 1: 1P and 2P excitation...adapted from Aggarwal et al…, Supplementary Figure 2.   Improved trafficking and localization through...fluorescence intensity from the inserted GFP. After characterizing iGluSnFR in vitro, showing that it is selectively...response than its predecessors. The detailed characterization of the new variants is available here. Upgrades...

  12. CRISPR-based Genome Editing Systems For Editing in E. coli

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    Blog Post
    ...plasmids are cured from the cell. Steps 1-1 and 1-2 can be done simultaneously. Image from Li et al., ...1) replaced the promoter of gRNA-pMB1 with PrhaB, 2) changed the replicon of pCas into a non-temperature-sensitive... pEcCas/pEcgRNA system in 4 E. coli K-12 strains, 2 E. coli B strains, 1 E. coli W strain, and 1 Tatumella...MUCICAT better, it is faster. Using MUCICAT only needs 2 newly constructed plasmids (pDonor and pQCascade) ...rounds of transformation in 30 days. Figure 2: (A) MUCICAT plasmids for multicopy integration. (...temperature-sensitive replicon for self-curing, an arabinose inducible lambda-red gene, and an IPTG-inducible...site of interest. The donor template is supplied separately. To use this system, you’d first introduce your...

  13. CasPEDIA: A Functional Classification of Cas Enzymes

    Type
    Blog Post
    ...systems: a burst of class 2 and derived variants. Nature Reviews Microbiology, 18(2), 67–83. https://doi.org...resource that provides summary information on Class 2 Cas enzymes. The information is organized in wiki ...this content and much, much more.     Fig. 2 – List of resources and properties for a Cas entry...Database: A Functional Classification System for Class 2 CRISPR-Cas Enzymes [Preprint]. Biochemistry, Biophysics... your hits, you can easily sort them by other parameters, such as E-value for protein sequence searches...

  14. Easi-CRISPR: Generating Knock-In and Conditional Mouse Models

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    Blog Post
    ...of conventional gene targeting is lowered to about 2-3 months. But certain models remain more difficult...included in the final ssDNA template. Figure 2: Overview of ivTRT to generate an ssDNA repair template... floxed conditional allele requires you to target 2 loxP sites to intronic regions 0.5 - 0.8 kb apart....Typically, Easi-CRISPR works best with insertions of 2 kb or less. Luckily, most of the commonly used cassettes... induction systems (ttA or rtTA) are between 1 to 2 kb in length. There are plenty of Addgene plasmid ...create the ssDNA construct as described in Figure 2.  You can also use Addgene plasmids to purify Cas9...ribonucleoprotein (RNP) complexes of Cas9 protein and the separated guide RNA components (crRNA and tracrRNA) for ...

  15. Hassle-free 96-well Format Epitope Tagging Using Cas9 Ribonucleoprotein

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    Blog Post
    ...purify IVT sgRNA in the lab. Figure 2: Cas9 RNP complexed with 2-part cr:tracrRNA tags genes more effectively...into stem cells. Cells are allowed to recover for 2-5 days and then analyzed for tagging efficiency using...IVT sgRNA) approach (Dewari et al. 2018 and Figure 2). Not only is the cr:tracrRNA approach highly efficient...cell suspension, transfect and let cells recover for 2-5 days.Note: We use the Nucleofection method for delivery...success was ~%50. If you get low knock-in efficiency (<2% positive by ICC for the tag) or do not see any cells...PMID: 28096221. PubMed Central PMCID: PMC5312033. 2. Dewari PS, Southgate B, Mccarten K, Monogarov G, ...assembling csRNPs in a 96-well plate format and tested parallel tag knock-in for 96 different genes in a single...

  16. Viral Vectors 101: AAV Variables That Matter

    Type
    Blog Post
    ...testing your AAV.  Figure 2: Comparison of AAV serotypes 1, 2, 5, 6, 8, and 9 in two different ...Yamamori, T. (2015). Comparative analyses of adeno-associated viral vector serotypes 1, 2, 5, 8 and 9 in marmoset...) your experimental conditions and measurements; (2) the cells you’re targeting; (3) how much expression... Tropism and Axonal Transport of AAV Serotypes 1, 2, 5, 6, 8 and 9 in the Mouse Brain. PLOS ONE, 8(9),... Comparison of four promoters. Gene Therapy, 28(1–2), 56–74. https://doi.org/10.1038/s41434-020-0169-1...site  (Kanaan et al., 2017), axonal transport  (Murlidharan et al., 2014), and postsynaptic transmission ... 1–41. https://doi.org/10.3390/cells12050785 Murlidharan, G., Samulski, R. J., & Asokan, A. (2014). Biology...

  17. Visualizing Your Plasmid Request Data

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    Blog Post
    ...with request data to the dashboard (Fig. 2).     Figure 2: Insert showing the upload function for... the date of downloading the CSV is shown (figure 2) and the dashboard displays the data in a standardized...microscopy (University of Amsterdam). He develops, characterizes, and uses genetically encoded fluorescent probes...

  18. Editor's Choice, October 2016

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    Blog Post
    ...Part 2: Building Community  My editor’s choice pick for this month is “Grad School Advice Part 2: Building...Options for CRISPR Genotyping Grad School Advice Part 2: Building Community Engineering the Plant Genome Using...graduate school classmates, Ben Vincent and Niroshi Senaratne. When we recorded, Ben and Niroshi, were finishing...

  19. CRISPR Between the Genes: How to Experiment with Enhancers and Epigenomics

    Type
    Blog Post
    ...for the 2+ million DHS sites. See below for further details. DNA is a 3D Fractal Figure 2: DNA compaction...: 1. Find Enhancers that have NANOG binding sites 2. Design a CRISPR screen to target and disrupt the ...NANOG enhancers 3. CRISPR out the enhancers from step 2. See which ESCs die or differentiate Step 3 reveals...expression for NANOG enhancers, we’re ready for step 2: design a CRISPR screen to disrupt these enhancers...to CRISPR out the enhancers we identified in step 2. The result is a heterogenous population of stem cells...determination was established with Bowtie2 using the parameters first described in Kearns et al.: bowtie2 -f ...

  20. 15 Hot Plasmids from 2017

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    Blog Post
    ...NGG)  Editing window ~2 nt  85175  pBK-EE-BE3  SpCas9 (NGG)  Editing window ~2 nt  85176  pBK-YE2-...quantum yield of 0.54) and mScarlet-H with M164H (2-fold improvement in photostability compared to mScarlet...SpCas9 base editors with editing windows as small as 1-2 nucleotides! To reduce off-target effects associated...pBK-YE2-BE3  SpCas9 (NGG)  Editing window ~2 nt  85177  pBK-YEE-BE3  SpCas9 (NGG)  Lower on-target activity...activity than BE3; editing window ~1-2 nt  87438  pET42b-HF-BE3  HF-Cas9 (NGG)  Very low off-target activity...Ladders Are Awesome Tools: They are comprised of 2 plasmids, pPSU1 and pPSU2, that can be amplified affordably...libraries The Gross Lab at UCSF recently deposited 2 Bacillus subtilis libraries with Addgene – the B. ...
Showing: 101 - 120 of 492 results