Frozen Yeast Competent Cells

Making Frozen Yeast Competent Cells

 

  1. Grow up intended stain (i.e. Y190) in appropriate media (YPAD) overnight.  (Approximately 1ml for every 100ml intended to inoculate the next day (i.e. 2.5 ml overnight to inoculate 250ml). If you are growing a yeast strain with a selectable plasmid. Grow in appropriate selection media about 20 ml for every 100ml intended to inoculate the next day. Some yeast strains in selection media may take 2-3 days to reach saturation. You should grow just to the point of saturation.

 

  1. Inoculate 250ml YPAD- this will be enough for about every seventy-five 100ul aliquots of frozen competent cells. Scale up as desired.  Let them grow to log phase (~0.7 OD). Doubling time for Y190 is about 3.5 hours. If you are inoculating a strain with a selectable marker inoculate to ~OD 0.3 so it goes through one doubling time. You don’t want too many doubling times as the may be lost due to non selection.

 

  1. Spin down cells (6,000 for 10 minutes) and wash in 0.4 volumes of starting volume (i.e 40 ml if you grew up 100ml culture) of a 100mM solution of LioAC.  (We do not wash in water as in other protocols and this allows us to skip the 30 min incubation step)

 

  1. Spin down cells again and wash in a 0.2 volumes starting volume with 100 mM LioAC.

 

 

  1. Spin down a final time and resuspend cells in 100mM LioAC with 15% DMSO or glycerol to a final volume of 0.03 of your starting volume. Note: Glycerol seems to work better for us.

 

  1. Aliquot 100ul shots in microfuge tubes and put cells into a cardboard box and allow to freeze slowly in –80C, unlike E. coli competent cells, flash freezing in liquid nitrogen will severely reduce their competency.

 

 

Solutions required:

1M Lithium Acetate (LioAC) 10.2 g in 100ml dH20 and  autoclave. Dilute to 100mM with dH2O for working solution.

Glycerol or DMSO