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We narrowed to 594 results for: cel.2

Showing: 461 - 480 of 594 results

  1. Back to Bacteria: CRISPR gRNA Multiplexing Using tRNAs

    Type
    Blog Post
    ...3570-5. doi: 10.1073/pnas.1420294112. Epub 2015 Mar 2. PubMed. Find the plasmids from this publication ...toolkit in human cells. Nissim L, Perli SD, Fridkin A, Perez-Pinera P, Lu TK. Mol Cell. 2014 May 22;54(...advantageous to instead use a preexisting, conserved cellular machinery to process gRNAs. Hijacking tRNA processing...tRNAs are among the most abundant molecules in a cell, this system is clearly also robust enough to ensure...54(4):698-710. doi: 10.1016/j.molcel.2014.04.022. Epub 2014 May 15. PubMed.  Find the plasmids from this...

  2. CRISPR/Cas9 FAQs Answered!

    Type
    Blog Post
    ...pluripotent stem cells differing exclusively at two early onset Parkinson Point mutations. Cell, 146(2), 318–331...extraction solution, runs the protocol, then uses around 2–4 µl in each 50 µl PCR reaction. The gDNA extracted...find both single-allelic and bi-allelic cells. Single-allelic cells usually make up the majority in culture...allows you to sort for GFP-positive cells and to enrich for those cells that were positively transfected....Double-Strand Break Repair in Mammalian Cells. Molecular and Cellular Biology, 18(1), 93–101. https://doi....genome editing specificity. Cell, 154(6), 1380–1389. https://doi.org/10.1016/j.cell.2013.08.021  Ran, F. ...CAS-Mediated Genome Engineering. Cell, 153(4), 910–918. https://doi.org/10.1016/j.cell.2013.04.025  Yang, H....

  3. Why and how to start studying chromatin (with Addgene's help)

    Type
    Blog Post
    ...JS, Legube G, Miller KM Genes Dev. 2015 Jan 15;29(2):197-211. DNA Cross-Bridging Shapes a Single Nucleus... R, Schmalhorst PS, Jude JG, Zuber J, Gerlich DW Cell. 2017 Aug 24;170(5):956-972.e23 Screen identifies... R, Schmalhorst PS, Jude JG, Zuber J, Gerlich DW Cell. 2017 Aug 24;170(5):956-972.e23 Rapid and phosphoinositol-dependent... Rando OJ, Xue Y, Swiderek K, Kuo A, Crabtree GR Cell. 1998 Nov 25. 95(5):625-36. An architectural role..., Ensminger AW, Fox AH, Chess A, Lawrence JB Mol Cell. 2009 Mar 27;33(6):717-26. Reporter-nanobody fusions...

  4. 27 Hot Plasmids from 2016

    Type
    Blog Post
    ...backbone, which expresses the gRNA from a Drosophila U6:2 promoter and Cas9 from the actin 5C promoter. Addgene...backbones for lentivirus LV 1-5 and retrovirus RV 2-5 were constructed. The authors then used GMAP assembly...engineering (MAGE) (1). MAGE uses recombineering (2) to simultaneously incorporate multiple single-strand...number of tandemly expressed gRNAs respectively) and 2) the size of the deletion. Smaller deletions generally... HEK293FT cells expressing a single gRNA targeting a single gene. The gRNA expressing cells were then ...plasmid to generate a population of mutant cells. The mutant cells were treated with WNV at a dose and duration...regulate the ability of WNV to induce cell death in infected cells (SEL1L, UBE2J1, EMC3, EMC2, DERL2, UBE2G2...

  5. Sharing is Caring: But How to Distribute Open Hardware?

    Type
    Blog Post
    ...-priced commercial devices.     Fig. 2: Workflow for open software (left) vs. open hardware (right...quantitatively evaluating antibody staining, or counting cells are now routine, open-source software projects that...portable device for studying plankton using flow cells, has become an established alternative to high-priced... and blueprints has the potential not only to accelerate scientific progress, but also to enable science...

  6. Plasmids 101: Sequence and Ligation Independent Cloning (SLIC)

    Type
    Blog Post
    ..., but at a lower efficiency (~20%). Figure 2: Schematic of multicomponent assembly with SLIC. Multiple...bacteria, so it can be transformed as-is into cells. The cells then get to work fixing up the “damage” and...nicked plasmid together long enough to get it into cells and let them do the repair work. The key is T4 DNA...

  7. Summer SciComm: Preprints

    Type
    Blog Post
    ... scientific communication ecosystem Figure 2: The scientific ecosystem, enriched by open access...from peer-reviewed publishing in a journal.  Accelerating scientific communication Preprints have been...Figure 1: Stand aside, CERN — it's a new age of accelerating science! Photo courtesy of Henry Mühlpfordt,...Lacy was also employed by the American Society for Cell Biology in a position working with preprints in ...

  8. How to Deposit Your Plasmids with Addgene

    Type
    Blog Post
    ...two options to begin your plasmid deposit. Figure 2: Choose to submit plasmid(s) online or by using a ...format. Figure 6: Add sequence, maps, and files. Step 2: Gene and Insert *Note: This page will not appear ...for a lentiviral vector to be used in mammalian cells, check Lentiviral and Mammalian Expression). This...

  9. Hot Plasmids and Viral Preps - March 2021

    Type
    Blog Post
    ...endothelial cell-pericyte, neuron-astrocyte, and cancer-stromal cell pairs.   Figure 2: Schematic...Winslow’s lab to label cells after cell-cell interactions. Using G-baToN, “sender” cells express GFP on the... and “receiver” cells express an anti-GFP nanobody on the cell surface. When the cells interact, GFP is...the receiver cell, and the receiver cell turns green. This system can be used in many cell types including...engineering with single-pot assembly Tracking cell-cell interactions New auxin inducible degron system....brgcm3sw     New system developed to track cell-cell interactions By Jennifer Tsang GFP-based Touching...of the interaction between the sender cell and the receiver cell. Image from Tang et al., 2020.   ...

  10. General Transfection

    Type
    Protocol
    ... solution can be stored at 4 °C for up to 2 months. After 2 months, discard the tube and thaw a new working... transfected using 1:1, 1:2, 1:3 and 1:6 µg of pRosetta :µg of PEI. The 1:2 and 1:3 ratios provided high...Plate 1x10 6 cells in a 75 cm 2 flask in a volume of 15 mL. Wednesday: Plate 1x10 6 cells in a T75 flask... transfect cells with a fluorescent plasmid using a variety of ratios. Check the cells 1–2 days after ...optimized for viral production) Day 1: Transfect Cells Day 2 (am): 18 h post transfection - Remove media, ...tissue culture plates. Incubate the cells at 37 °C, 5% CO 2 for ~20 h. Gently aspirate media, add 10 mL...Biological Safety Cabinet 0.5–10 µL single channel pipette 2–20 µL single channel pipette 20–200 µL single channel...

  11. Mouse Modeling, Part 1: Genetically Engineered Mice

    Type
    Blog Post
    ...covered in more detail in part 2 of this blog series) controls the cell types in which the conditional...modified zygotes or embryonic stem cells. Modified embryonic stem cells are injected into a blastocyst which...engineered cells. Similar to transgenic mice, knockout mice are engineered during the embryonic stem cell stage...not all cells in the blastocyst were successfully edited by the injection of embryonic stem cells. Also,... interest into a zygote, or into embryonic stem cells that are then injected into a blastocyst. There ...of interest into a zygote or into embryonic stem cells. Non-homologous recombination incorporates this ...targeting vector is electroporated into embryonic stem cells and this will insert the gene at a specific site...

  12. Tips for Titering Your Lentiviral Preps

    Type
    Blog Post
    ...protocols 4.4 (2009): 495-505. PubMed PMID: 19300443. 2. Geraerts, Martine, et al. "Comparison of lentiviral...can actually infect cells. These assays typically involve infecting a target cell line with your virus...distinguish between a cell that has had one viral particle integrated into its genome and a cell that has had ...arrived; you’ve painstakingly cared for your packaging cell line, prepped your DNA, transfected and harvested...move ahead with your infection and make your stable cell line. While we’ve all experienced the pressure to...viral copies that have integrated into the target cell’s genome. Physical titer The two most common assays...functional titer uses FACS to count the number of target cells that are positive for vector encoded transgene expression...

  13. Summer SciComm Series: A PhD in Science Communication

    Type
    Blog Post
    ...perceived climate change risks. Nature Climate Change, 2(10), 732-735. doi:10.1038/nclimate1547 Kunda, Z. (...spend the afternoon lovingly tending to the stem cells I was using in an experiment, then spend the evening...

  14. 3 Challenges in Plant Synthetic Biology

    Type
    Blog Post
    ...and open a new conversation about GMOs.  Challenge #2: Technical obstacles to plant synthetic biology In...challenging to engineer a specific function in these multicellular organisms. However, the rewards are great if...

  15. Lentivirus ddPCR Titration

    Type
    Protocol
    ...Activation 95 10 2 1 Denaturation 94 0.5 2 40 Annealing/Extension 60 1 2 40 Enzyme Deactivation 98 10 2 1 Hold ...copies of RPP30 in diploid cells, thus the reason for multiplying by 2. $$V = 2*{copies\ RRE \over copies...diluted 2-fold serially, the concentration of RRE positive droplets should decrease by a factor of 2 across...each well. Detach cells by incubating with 200 µL TrypLE for 1–2 min. Resuspend cells in 500 µL DMEM complete...Lentivirus is generally considered biosafety level 2+. Please ensure that you are in compliance with your...channel pipette 200–1000 µL single channel pipette 2–50 µL multichannel pipette 20–200 µL multichannel ... DG8 cartridge into the cartridge holder. Using a 2–50 µL multichannel pipet, load 20 µL of the reaction...

  16. Plasmids 101: The protein expression toolbox

    Type
    Blog Post
    ... the transcriptional level.     Fig. 2 Tetracycline on/off promoters and modes of action ...force to control protein expression in mammalian cells: easy to use biological tools are abundant! In this...tag prior to introduction of the construct into cells. However, if you would like tunable control of an...

  17. Announcing the Winners of the 2021 Michael Davidson and Roger Tsien Commemorative Conference Awards

    Type
    Blog Post
    ...processes that happen inside the cell,” says Pedre Pérez. Figure 2: Micrographs of the biosensor ...sensitive GFP2 (roGFP2)-based biosensors for live cells. His work focuses on the enzyme hydropersulfide-...biosensor expressed in the human cell line, Hep3B. From left to right: Mitotracker Red, GFP for the biosensor,...studies opioid use disorder. Her goal is to identify cell populations in the brain that contribute to the ...will explore the connectivity patterns of these cells and the functional contribution to a variety of ...administration and withdrawal to explore how these cells may encode the negative affect associated with drug...library of mutant sensors expressed in a mammalian cell line engineered to be electrically excitable. The...

  18. Antibodies 101: Affinity Reagents

    Type
    Blog Post
    ...Antibody Fragments. Antibodies (Basel). 2019 Apr 9;8(2):28. doi: 10.3390/antib8020028. PMID: 31544834; PMCID...in transfected eukaryotic cells, allowing for them to be used as intracellular inhibitors.  Aptamers are...the kids say) affinity reagents deserved to be celebrated, there are many other amazing affinity reagents...small enough to be expressed in bacterial and other cell systems. Since the majority of these alternative...the constant region. Nonspecific binding, caused cell receptors for the constant region, is also reduced..., peptides, amino acids, drugs, metal ions, and cells. They can be used either with or as fluorophores... may sound. Validated antibodies are still an excellent option for many applications, and frankly, if ...

  19. RNA Extraction Without A Kit

    Type
    Blog Post
    ...156–159. https://doi.org/10.1016/0003-2697(87)90021-2 Chomczynski P, Sacchi N (2006) The single-step method...are commonly found in cells, and without something to block RNase activity upon cell lysis, RNA is degraded...RNases such as RNaseZAP. When harvesting tissues, cells, plants, fungi, or bacteria, keep samples cold and...before RNases had a chance to break down RNA upon cell lysis.  In the late 1970’s, Chirgwin and colleagues...keeping your samples cold until homogenization and cell lysis. This can be challenging depending on your...developed ways to “stabilize” RNA within tissues before cell lysis. These products, namely RNAlater®  from Thermo...solutions that work by inhibiting RNase activity within cells or tissues- they don’t actually chemically stabilize...

  20. Lentivirus Production

    Type
    Protocol
    ... solution can be stored at 4 °C for up to 2 months. After 2 months, discard the tube and thaw a new working...μg total DNA to μg PEI ratios of 1:1, 1:2, 1:3 and 1:6. The 1:2 and 1:3 total DNA:PEI μg ratios provided... transfect cells with a fluorescent plasmid using a variety of ratios. Check the cells 1-2 days after ...Transfect packaging cells Day 2 (am): 18 h post-transfection. Remove media, replace with fresh media. ...Biological Safety Cabinet 0.5–10 µL single channel pipette 2–20 µL single channel pipette 20–200 µL single channel... 200–1000 µL single channel pipette Ice bucket CO 2 incubator Pipet controller Hazardous waste container..., 5% CO 2 for ~20 h. Prepare a mixture of the three transfection plasmids into OptiPro SFM: Reagent Amount...
Showing: 461 - 480 of 594 results