Quint Lab:Short version ß-galactosidase assay

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Short version ß-galactosidase assay Try to be fast!!! Everything at the same time – as good as possible 2 ml over night culture (X-Gal plate): 3 + x independent clones prepare ONPG (4 mg/ml Z-buffer) dilute over night culture to OD 0.2 harvest if OD600 is 0.6 – 1.0 resuspend in same amount Z-buffer → measure OD600 (2 ml tubes!) keep on ice take 200 µl (try out before) cells + 800 µl Z-buffer containing ß-Mercaptoethanol + 25 µl 0.1% SDS + 50 µl Chloroform → mix 150 @ 30°C, waterbath add 200 µl ONPG → mix 30°C, waterbath for 20 min (try out before – till it is yellow) centrifuge 10 min top speed measure OD420 of the SN calculate Miller units