Yeast transformation stationary (LKL 2014 protocol)

From LKL lab Yeast 2014 –transformation eff of >50,000 transformants/ ug DNA

Spin down 1.5 mL of cells grown overnight at 30 °C in YPD, 15 s at full speed (discard spnt).

to the pellet:

5 µL recently boiled 10 mg/ml carrier DNA,

2–5 µL pRS316 DNA (100–700 ng)

500 µL (PLTED) solution were added.

Mix

+ 56 µL DMSO was added,

Mix

incubated at 30 °C for 15 min.

42°C for 20 min,

spin for 15 s, and the resulting cell pellets were

resuspended in 1ml YPD.

30C for 40 min

Spin down, resuspend in 500ul water and plate to selective media

PLETD                                                                    5X                                           10X

800 µL 50% PEG−4000                                          4 ml                                        8 ml

100 µL 1 M LiAc                                                   500 ul                                        1 ml

20 µL 50mM EDTA                                               100 ul                                     200 ul

10 µL 1 M Tris (pH 7.5)                                         50 ul                                      100 ul

80 µl of 1 M DTT (100mM DTT) 400 ul                                      800 ul

Enough PLTE was prepared for all transformations to be performed plus at least one more.