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SDS-Polyacrylamide solutions and buffers

 

 

 

 

 


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 30C, 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 (~50l)

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

Mix thoroughly by vortexing/pipetting

Incubate 1h at 37C, 250rpm

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

Freeze samples at -20C

Thaw

Vortex

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

Add 250l Buffer P2

Etc.. follow QIAGEN protocol

Elute DNA in 30l of H2O

Use 20l of eluted DNA to transform 200l 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.