Yeast Culture
1. Grow a yeast culture in 5-10 ml to an absorbance of about 1.5 at O.D.600.
(Want 6-8 dblings ovn (Ken) or 10 dblings (Ted);or use
Chris's anal-retentive method shown below. Up to 2.0 in YPD before they derepress. Keep to O.D.600
= 0.7 for the strains that easily derepress (e.g. multicopy ADR1).
2. Pellet the cells and discard the supernatant.
3. Wash the cells with 0.7 ml of solution, either buffer +
glycerol (e.g. Z-buffer + glycerol or ADH buffer + glycerol) or water. Pellet the cells, discard the
supernatant, and freeze the cells at -70¡C or dry ice then -70.
Spring 2004, We are now using Invitrogen non-denature
gels for in-gel ADH activity assays.
Getting a culture to be at the correct OD, when you
want it:
1. Measure the cell number or OD600 of a culture of your strain as it is actively growing and determine the doubling time using this formula:
[log10 (Nt/N0)] / 0.3 = g
doubling time = t/g
N0 = # of cells or OD600 at start
Nt = # of cells or OD600 at the end
t = time cultured
2. Put a single colony from a plate into 5 ml YPD 5% glucose. Shake 30o all day
¥ at least 6 hours, for sick strains 12-24
3. Measure OD600
¥
Read in the 0.05-0.7 range, so dilute if necessary
4. Determine the amount of all-day culture to add to a larger, overnight culture using this formula:
N0 = Nt/2g
N0 = OD600 at which to start
Nt = OD600 that you want at the end
g = number of generations the culture will go through before harvesting
¥
example:
You
want the culture to grow overnight from 7:00 p.m. - 8:00 a.m.
You want a final OD600 of 0.7
If
your strain is wildtype W303 in YPD 5% glucose, it has a 1.3 hour doubling time
7:00
p.m. - 8:00 a.m. is 13 hours, so g = 10
N0 = Nt/2g =
0.7/210
Start
the culture at OD600 = 6.8 x 10-4
If
the all-day culture is OD600=1 put 68 µl into 100 mls of medium or
6.8µl into 10 mls and shake 30o for
13 hours. Voila.