酵母质粒的提取

Pick colonies into 0.5ml of SD-Leu (or other appropriate SD medium)

Vortex for 1min

Leave to grow O/N for 18-24h at 30℃, 230-250rpm (best in 5ml bijou)

Spin yeast culture at 13,000rpm for 5 min (microfuge)

Pour off supernatant carefully and resuspend the pellet in residual liquid (~50µl)

Add 10µl Lyticase (Sigma L2524 at 5 Units/µl in TE; aliquots stored in at -20℃)

Mix thoroughly by vortexing/pipetting

Incubate 1h at 37℃, 250rpm

Add 10µl of 20% SDS and vortex for 1min

Freeze samples at -20℃

Thaw

Vortex

Start QIAGEN miniprep protocol by adding 180µl of Buffer P1 to obtain a final volume of 250µl

Add 250µl Buffer P2

Etc.. follow QIAGEN protocol

Elute DNA in 30µl of H2O

Use 20µl of eluted DNA to transform 200µl competent XL1-Blues

Plate on LB/Amp, grow up from colonies and miniprep.

Alternatively: Inoculate direct to 5ml LB/Amp O/N and on to LB/Amp plates 50:50. Isolate plasmid using QIAGEN minipreps.