Protocols
1) Thaw the YKO collection. Our YKO collection is arrayed in 96 well format.
2) Inoculate each YKO strain into ~150 µl of liquid YPD + 200 µg/ml
of G418 for 1 to 2 days.
3) Pin each YKO strain from the liquid culture onto YPD plates containing
G418 (200 µg/ml) as individual 5-mm patches. The YKO strains
are usually plated in 96 well format onto rectangular Nunc Omnitrays.
Make sure every yeast strain is pinned well. It is worthwhile to pin
every 96 well plate of YKO strains onto two separate plates. For YKO
strains that were not pinned well or not well represented, more cells
could be obtained from the second plate to make the pool more balanced
with respect to representation.
4) Incubate at 30°C for 3 days.
5) Scrape all YKO strains into ~300 ml of 15% glycerol. This is usually
done in a pre-autoclaved 1-liter beaker containing a stir bar.
6) Adjust OD600 of the YKO pools to 15 OD600/ml with 15% glycerol. For the MATa haploid YKO pool, this required an addition of ~500
ml of 15%
glycerol.
7) Aliquot the yeast pool as 1- to 2- ml aliquots into cryogenic
vials. While aliquoting, the beaker is placed on top of a magnetic
stirrer
to constantly
stir the yeast pools ensuring a well-mixed pool.
8) Freeze at –70¾C.