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  • Results: Both GFP-RBM6 WT and GFP-RBM6 UV Mutant Are Recruited To Laser Micro-Irradiated Sites With Similar Kinetics

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    fmachour9316
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    The document discusses experiments involving RBM6 recruitment and KCr after VP16 treatment in various cell lines. It notes that wild type and UV mutant GFP-RBM6 were recruited to laser micro-irradiated sites with similar kinetics in U2OS cells. It also mentions plating Hras wt and KO MCF10A cells, as well as U2OS cells, for experiments involving VP16 treatment and subsequent RNA extraction from MCF10A cell lines. Notes indicate that cells should be washed after Hoechst staining before imaging and that less DNA should be used for future RBM6 recruitment experiments due to high expression.

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    1.10.18

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    The document discusses experiments involving RBM6 recruitment and KCr after VP16 treatment in various cell lines. It notes that wild type and UV mutant GFP-RBM6 were recruited to laser micro-irradiated sites with similar kinetics in U2OS cells. It also mentions plating Hras wt and KO MCF10A cells, as well as U2OS cells, for experiments involving VP16 treatment and subsequent RNA extraction from MCF10A cell lines. Notes indicate that cells should be washed after Hoechst staining before imaging and that less DNA should be used for future RBM6 recruitment experiments due to high expression.

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    6 views1 page

    Results: Both GFP-RBM6 WT and GFP-RBM6 UV Mutant Are Recruited To Laser Micro-Irradiated Sites With Similar Kinetics

    Uploaded by

    fmachour9316
    The document discusses experiments involving RBM6 recruitment and KCr after VP16 treatment in various cell lines. It notes that wild type and UV mutant GFP-RBM6 were recruited to laser micro-irradiated sites with similar kinetics in U2OS cells. It also mentions plating Hras wt and KO MCF10A cells, as well as U2OS cells, for experiments involving VP16 treatment and subsequent RNA extraction from MCF10A cell lines. Notes indicate that cells should be washed after Hoechst staining before imaging and that less DNA should be used for future RBM6 recruitment experiments due to high expression.

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    © All Rights Reserved
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    of 1

    1.10.

    18

     RBM6 mutants recruitment in U2OS:


    o Add 1:1000 Hoechst (2ul in 2ml) to cells for 10 minutes and incubate at 37C 5% CO2.
    o Discard media + Hoechst and add 2ml L15 media.

    Results: Both GFP-RBM6 WT and GFP-RBM6 UV mutant are recruited to laser micro-
    irradiated sites with similar kinetics.

     KCr after VP16: in MCF10A Hras cells and U2OS cells:


    o Plated Hras wt and Hras CDYL1 KO cells at ~40% confluency in 6 6cm plates.
    o Plated U2Os cells ~40% confluncy in 3X 6 6cm plates (18 plates in total).
     Plated MCF10A Hras wt and Hras RBM6 KO (nickase) in 35mm plates at ~50% confluency for
    RNA extraction the following day.

    *** NOTES:

     I should wash the cells after addition of Hoechst before adding L15 media
     Next time for RBM6 recruitment: I should use less DNA – expression was too high

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