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We narrowed to 594 results for: cel.2

Showing: 521 - 540 of 594 results

  1. New CRISPR Tools: Cas7-11 and PASTE

    Type
    Blog Post
    ...rate of 10-55% - the PASTE system.     Fig. 2: Schematic of programmable gene insertion with PASTE...no detectable effect on cell viability – meaning there was no measurable cell toxicity – using Cas7-11...such as mouse embryonic stem cells and glioblastoma cells. PASTE Development and discovery The first ...observed in non-dividing cells, meaning that this system wasn’t dependent on the cell’s intrinsic machinery...turning potential into impact. In molecular and cellular biology, this happens through the development ...exclusively on this space — developing tools for cellular targeting. Lately, they’ve been working on two... shRNA and Cas13 systems in a range of mammalian cell lines. However, in those same comparisons, the AbuGoot...

  2. Plasmids 101: Multicistronic Vectors

    Type
    Blog Post
    ...al (Nature. 1988 Jul 28;334(6180):320-5.) PubMed. 2. A segment of the 5' nontranslated region of encephalomyocarditis...them. Why Use multicistronic vectors? Detecting cells that are expressing your gene, especially if you...reporters allow you to easily screen or select for cells that are expressing your gene of interest at high...promoter, multicistronic plasmids ensure that any cells that are positive for your marker should also be...anytime you want to express multiple genes in the same cell. Although it is possible to drive co-expression ...promotes translation of the viral genome in eukaryotic cells.1,2 Since then, a variety of IRES sequences have...discovered - many from viruses, but also some from cellular mRNAs. What they all have in common is the ability...

  3. New Optimized Genome-wide CRISPRko, CRISPRi, and CRISPRa Libraries

    Type
    Blog Post
    ... PMID: 25184501. PubMed Central PMCID: MC4262738. 2. Doench, John G., et al. "Optimized sgRNA design to...when trying to screen a limited number of cells like primary cells or in vivo. Recently, the labs of David... cultured cells as well as more difficult conditions such as model systems and primary cells. References...with both A375 (melanoma) and AT29 (colon cancer) cells and assessed the libraries performance via the depletion...important criterion for probing a limited number of cells. Get the Brunello CRISPRko sgRNA library as pooled...subject to an increase in dsDNA breaks that can cause cell death. Thus, CRISPR inhibition (CRISPRi) libraries...study screening for vemurafenib resistance in A375 cells. The SAM system (CRISPR/Cas9 Synergistic Activation...

  4. What is Polymerase Chain Reaction (PCR)

    Type
    Protocol
    ...PCR tube Ice Bucket 2 μL Template DNA (10 ng-500 ng) 5 μl 10X Taq buffer with MgCl 2 1 μl dNTP mix (10 ... reagents on ice): 2 μL Template DNA (10 ng-500 ng) 5 μl 10X Taq buffer with MgCl 2 1 μl dNTP mix (10 ...normally sterile dH 2 O. To make a 100uM stock of any primer, add a number of µl of dH 2 O equal to the number...annealing temperature step-wise by 1-2°C. The rate of DNA synthesis is ~1-2 kb/min. The extension time can ...work adequately. Divalent cations such as Mg 2+ and Mn 2+ stabilize the buffer solution. These cations...(PCR). Basic PCR Program Initial Denaturation for 2 minutes at 94°C: This initiation step heats the double...target DNA strand accurately and rapidly. Repeat steps 2-4 25-30 times. Final Extension for 5 minutes at 72...

  5. Kit Free RNA Extraction

    Type
    Protocol
    ...RNAzol®, QIAzol® (for Protocol Option #2) Water-saturated Phenol 2 M Sodium Acetate pH 4 Chloroform/Isoamyl...to the Option #2 - TRIzol® Protocol section below. Homogenize or lyse tissues or cells in Solution D. ...those in -80°C. Option #2 - TRIzol® Protocol Homogenize or lyse tissues or cells in TRIzol® or a similar....5% (wt/vol) N-laurosylsarcosine (Sarkosyl) 0.1 M 2-mercaptoethanol TRIzol® or similar product such as...following sequentially to 1 mL of lysate: Add 0.1 mL of 2 M sodium acetate (pH 4.0), mix thoroughly by inversion...by hand for 10 seconds. Incubate the sample(s) for 2-3 minutes on ice and centrifuge for 15 minutes at ... D per 100 mg of cells. For cultured cells: use 1 mL of Solution D per 1 X 10 7 cells. Allow sample(s)...

  6. Design Tips for Prime Editing

    Type
    Blog Post
    ...inhibit editing (Anzalone et al., 2019). Figure 2: Starting lengths for optimizing your pegRNA primer...editing systems by manipulating cellular determinants of editing outcomes. Cell, 184(22), 5635-5652.e29. https...Guide RNA and Target DNA. Cell, 156(5), 935–949. https://doi.org/10.1016/j.cell.2014.02.001 Yan, J., Oyler-Castrillo...type of edit, genomic context, delivery strategy, cell type, and goal of the experiment. Now, onto the ...nicking of the unedited strand and encourage the cell to use the edited strand as a template during DNA...also allows certain unintended edits to escape cellular surveillance. To limit unintended incorporation...https://doi.org/10.1016/j.cell.2021.09.018 Nelson, J. W., Randolph, P. B., Shen, S. P., Everette, K. A...

  7. Degrading DNA with Cascade-Cas3

    Type
    Blog Post
    ...systems utilize with type I-F Cascades. Figure 2: Cascade complex from CRISPR type I-E. Created with...unwinding steps. Molecular Cell, 70(3), 385-394.e3. https://doi.org/10.1016/j.molcel.2018.03.031 Xiao, Y., ...CRISPR Cascade. Molecular Cell, 83(5), 746-758.e5. https://doi.org/10.1016/j.molcel.2023.01.024 Morisaka, ...there isn’t a template strand left behind that the cell can use to repair. Figure 3: General overview...broad and unidirectional genome editing in human cells. Nature Communications, 10(1). https://doi.org/10.1038...CRISPR–Cas systems for genome engineering in human cells. Nature Biotechnology, 37(12), 1471–1477. https:...

  8. AAV Purification by Iodixanol Gradient Ultracentrifugation

    Type
    Protocol
    ...purification. Workflow Timeline Day 1: Purify Day 2: Buffer exchange and concentration Note: Both steps...7.4 1X PBS-MK buffer 100X Poloxamer 188 NaCl MgCl 2 KCl Centrifugal filter units (MWCO 100 kDa) Reagent...PBS-MK buffer Dissolve 5.84 g of NaCl, 26.3 mg of MgCl 2 and 14.91 mg of KCl in 1× PBS in a final volume of...at 4 °C. 1X PBS-MK buffer Dissolve 26.3 mg of MgCl 2 , and 14.91 mg of KCl in 1× PBS in a final volume ...(C) (formulation buffer) Add 5 mL of Buffer B and 2 mL of 5 M NaCl to 43 mL PBS Procedure Preparation ...need more time, you can alternatively centrifuge for 2 h at 200,000 x g at 18 °C. Carefully take the QuickSeal...interface of the 60% and 40% gradient (see Figure 2) with an 18 ga needle. Place the first microcentrifuge...

  9. Tips from the Repository Trenches: Using Barcodes to Track Samples

    Type
    Blog Post
    ...that numbers are not switched around or misread.  2. Track the container - It is just as important to ... reader and an Excel spreadsheet. Start by scanning the sample barcodes into the cells in the same order...using an electric charge to help the DNA enter the cells. Some samples require a reduced antibiotic concentration...concentration or need to be transformed into different cells than were originally requested. Most of our samples...tubes and use this ID to name the corresponding Excel sheet.3. Be specific  - When assigning a physical...storage process - from deposited DNA to transformed cells and finally to bacterial glycerol stocks that are...

  10. Corralling and Cornering the Goldilocks Antibody

    Type
    Blog Post
    ...non-specific binding in our IHC condition.   Fig. 2: Schematic of the goldilocks antibody selection process... Innovative Neurotechnologies (BRAIN) Initiative Cell Census Network, the Wu lab will be screening over...

  13. Save Time with Transient Plant Leaf Transformations

    Type
    Blog Post
    ...GUS-staining is a very useful visual reporter (Figure 2). The GUS enzyme cleaves a substrate, which will then...roots, as functional chloroplasts and leaf specific cell types are missing in roots. The development of transgenic...transformation” usual means that neither a stable cell line or plant is regenerated from the transformed...with the gene of interest is transferred into the cells. But for C. roseus, Agrobacterium infiltration into...fused to a protein of interest to elucidate the subcellular localization of your protein of interest. Find...Intrinsic GUS-like activities in seed plants." Plant cell reports 9.1 (1990): 1-5. PubMed PMID: 24226366. ...

  14. To Codon Optimize or Not: That is the Question

    Type
    Blog Post
    ...Hoogsten base pairs and wobble pairs.  Figure 2: (A) The codon wheel depicts the 64 codons that encodes...mammalian cells. This was the first codon optimized membrane protein expressed in mammalian cells. In this...sequence. That means there are 61 possible tRNAs. Cells however, may not express all 61 of these tRNAs and... are also optimized for expression in mammalian cells. For example, Henry Lester’s lab optimized the C...Caenorhabditis elegans GluCl ion channel genes for mammalian cells dramatically improves expression levels. Journal...

  15. Plasmids 101: Codon usage bias

    Type
    Blog Post
    ...PMID: 18478103. PubMed Central PMCID: PMC2364656. 2. Dittmar, Kimberly A., et al. "Selective charging ...reasons include: Metabolic pressures - it takes cellular resources to produce tRNAs that recognize different...mismatched translation rates and potential effects on cell growth, even hosts producing alternative tRNAs may...inclusion bodies that will generally segregate with the cell pellet during purification procedures. Even if you...a means to fine-tune gene expression." Molecular cell 59.2 (2015): 149-161. PubMed PMID: 26186290. PubMed...

  16. Course-Based Undergraduate Research Experiences (CUREs)

    Type
    Blog Post
    ...that view (Callahan et al., 2022).     Fig. 2: Students in an MDH CURE replaced select residues ...identifying new genes required for stem and progenitor cell maintenance in planarians in Developmental Biology... his teaching style, with the former offering excellent training in improvisation and the latter making...junction complex is required for stem and progenitor cell maintenance in planarians. Developmental Biology...

  17. Transferable Skills Guide: Career Planning Resources

    Type
    Blog Post
    ...American Society for Cell Biology (ASCB) or the American Chemical Society (ACS) are just 2 examples of organizations...for the position. This post on Business Insider nicely summarizes good job fair practice. Career pages..., there is an etiquette that you should follow, nicely summarized in this post on Forbes.com, and here...

  18. Protocols for Molecular Biology, Plasmid Cloning, and Viral Preps

    Type
    Protocol
    ...BSL-1 and BSL-2 Labs Learn how to best protect yourself when working in BSL-1 and BSL-2 labs Watch the...Two (BSL-1 and BSL-2) Safety measures for laboratories operating at BSL-1 and BSL-2 Watch the Video! Water...Generating Stable Cell Lines with Lentivirus Genomically integrate your DNA sequence into a cell line Isolating...Isolating a Monoclonal Cell Population by Limiting Dilution Generate monoclonal cell lines from a polyclonal...polyclonal pool of stable cells AAV Production in HEK293 Cells Produce adeno-associated virus to deliver your...Extraction Without a Kit Extract RNA from tissues or cells without a commercial kit Plasmid Cloning Protocols...Transfection Introduce plasmid DNA to mammalian cells Lentivirus Production Produce lentivirus with a ...

  19. Protocol - Bacterial Transformation

    Type
    Protocol
    ...each transformation tube by placing the bottom 1/2 to 2/3 of the tube into a 42°C water bath for 30-60 ...depending on the competent cells you are using). Put the tubes back on ice for 2 min. Add 250-1,000 μl LB...the competent cells in your hand instead of on ice Reduce step 4 from 20 - 30 mins to 2 mins on ice before...commonly referred to as 'competent cells.' Many companies sell competent cells, which come frozen and are prepared...that came with your competent cells. Pro-Tips Commercial competent cells range significantly in their ...SOC media Competent cells DNA you'd like to transform Procedure Take competent cells out of -80°C and thaw...use electro-competent cells. Instead of relying on the heat-shock to cause the cells to take up the DNA,...

  20. Antibody Validation Using the Indirect ELISA Method

    Type
    Protocol
    ...vapors. Workflow Timeline Day 1: Antigen Coating Day 2: Blocking Day 3: Primary antibody incubation Day 4...with 96-well plates 1–10 µL single channel pipette 2–20 µL single channel pipette 20–200 µL single channel...serial dilutions of the purified antigen as follows: 2 ng/µL : Add 100 µL of 20 ng/µL stock into 900 µL PBS...microfuge tube and vortex. 1 ng/µL : Add 450 µL of 2 ng/µL stock into 450 uL PBS in a microfuge tube and...37 °C for 30 min , or overnight at 4 °C . Section 2: Block the plate Prepare the wash buffer (0.05% Tween...incubate on a microplate shaker set to 400 rpm for 2 h at room temperature or overnight at 4 °C . Section...
Showing: 521 - 540 of 594 results