Quint Lab:Short version ß-galactosidase assay

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