Drop assay for S.pombe

Contents

1. Procedure
2. Notes

1. Procedure

1. Grow cells in YES media at 30°C with shaking to mid log phase.
2. Spin down the equivalent of 1 million cells (1600g 3 mins) and resuspend in 250μl of YES media (1ml of cells at OD600=0.5 = 10⁷ cells).
3. Pipette into the first tube of an 8 tube PCR strip.
4. Add 200μl of YES to the other 7 tubes.
5. Pipette 50μl from the first tube into the second, then 50μl from the second into the third etc to perform serial dilutions.
6. Using an agar plate that has been allowed to dry slightly (not so much as to have cracks) trace a grid underneath the plate to serve as a guide for the drops.
7. Pipette 5μl from each tube in a line across the agar plate. (pro-tip: if you start from the least concentrated tube you won’t have to keep changing pipette tip)
8. Let the plate sit on bench for 10-15 minutes drop side up to allow the liquid to soak into the agar. Take care not to move the plates around too much at this point so as not to smudge the drops.
9. Place in incubator as normal at 30°C.

2. Notes

Colonies should be large enough within 2-3 days