Title: ylic133 and ylic136 transformation with bem3d::NAT

26. Title: ylic133 and ylic136 transformation with bem3d::NAT#

26.1. Date#

04122020-09122020

To get the following mutants for SATAY:
- \(\Delta\)bem3 in the WT background
- \(\Delta\)bem3 in the \(\Delta\)nrp1 background

Yeast Transformation Protocol
Culture 10ml of from each strain from glycerol stock (the afternoon before today) in YPD+6xADE to avoid adenine deficiencies.
OD measurement -ylic133: 3.15 -ylic136: 1.24
I did not dilute them , so continue with this because they were in log phase. (In the morning they were not dense at all)
Amount of DNA added: 10ul -> 10uL*260ng/uL=2.6ug
I included one negative control per strain
Recovery time : 1h 20mins
Plate the whole culture in the selection plates: YPD+NAT+6xADE
- 1mL plated after recovery without any dilution.

High efficient transformation :)
The negative controls have some colonies, very localized in one place, which could mean that in that region there was not enough NAT. I spread NAT with glass beads.

Culture single colonies (6) per strain to freeze in glycerol stocks to check later their integration with PCR.
- Only 1 colony from the 6 colonies has densely grown after 24 hours in 30C incubation. I stored that one from each strain in glycerol stocks.
- I stored the plates with the transformants and the restreaking of dbem3dnrp1 single colonies to regrow them after January. (coming back from my holidays :D)