Plasmid isolation from yeast

 

 

 

(see Clontech YPH, p31)

 

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°C, 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°C)

 

Mix thoroughly by vortexing/pipetting

 

Incubate 1h at 37°C, 250rpm

 

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

 

Freeze samples at -20°C

 

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 H2 O

 

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.