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


 * 1) 2 ml over night culture (X-Gal plate): 3 + x independent clones
 * 2) prepare ONPG (4 mg/ml Z-buffer)
 * 3) dilute over night culture to OD 0.2
 * 4) harvest if OD600 is 0.6 – 1.0
 * 5) resuspend in same amount Z-buffer → measure OD600 (2 ml tubes!)
 * 6) keep on ice
 * 7) take 200 µl (try out before) cells + 800 µl Z-buffer containing ß-Mercaptoethanol
 * 8) + 25 µl 0.1% SDS + 50 µl Chloroform → mix
 * 9) 150 @ 30°C, waterbath
 * 10) add 200 µl ONPG → mix
 * 11) 30°C, waterbath for 20 min (try out before – till it is yellow)
 * 12) centrifuge 10 min top speed
 * 13) measure OD420 of the SN
 * 14) calculate Miller units