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.