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        Routine Splitting and freezing of cells

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        716

         

        1. Grow cells to subconfluence in a flask.

        2. Harvest as per normal and count.

        3. Spin down 5min 1.2K in benchtop. Resuspend at 1.0 X 106/ml in 10% DMSO in media. This is prepared from frozen aliquotss of DMSO (essential to use tissue culture grade eg. DMSO Sigma D-2650, aliquoted and frozen at -70�). Filter DMSO / media prior to resuspending cells in it.

        4. Add cells to Nunc cryotubes (1ml ea. Cat. 3-63401) and label with initials, experiment no. and cell name in pencil.

        5. Place in a Mr Frosty (Nalgene #5100-0001) warmed to room teperature, the outer container filled with isopropanonol to the indicated line and then put container into bottom of -80� freezer.

        6. Tranfer to LN2 next day and record position in cell database.

         

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