1. Grow 3 ml ON cultures in selective media.
2. Dilute the cells,
into 6 ml of fresh media, to an OD600; = 0.3 and grow the
cultures for 3h to an OD600 between 0.5 and 1.0 (assuming
107 cells per OD600)
3. Measure the
OD600; Take 1 ml samples of each culture and pellet the cells
in microfuge tubes.
4. Resuspend the
cells in 1 ml of Z buffer.
Z Buffer: 16.1
g. Sodium dibasic •7H2O
5.50 g. Sodium
monobasic • H2O
0.75 g. KCl
0.25 g. MgSO4•7H2O
Q.S. to 1 l. with
H2O.
2.7 µl. ß-mercaptoethanol
is added, per ml, just before use
5. Transfer the
cells to a glass tube and add 25 µl. of 0.1% SDS and
50µl. Chloroform to each sample.
6. Vortex for 1
min.
7. Preincubate
the samples for 5 min. at 28°C.
8. Add 200 µl.
of 4 mg/ml ONPG in 100 mM Na phosphate pH 7 to each sample
and mix; Record this time as TIME 0.
9. Incubate at
28°C.
Since the amounts
of ß-galactosidase will vary between samples the time
that it takes for the yellow color to develop will also vary
(up to 2h.); therefore, each tube has to be watched carefully.
10. When a sample
turns yellow add 0.5 ml. 1M Sodium carbonate to stop the reaction.
Record the time you stopped the reaction as TIME t.
11. Pellet the
cell debris in a microfuge (2 min.) and carefully remove the
supernatent without distubing the loose pellet or removing
any Chloroform.
12. Measure the
OD420 for each sample and calculate the units as follows.
OD420
ß gal units
? 1000 ¥ æææææææ
with ?t ??TIME t-TIME 0 (in min.)º
?t v (OD600) and
v ??sample volume (usually 1 ml)
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