Smolke:Protocols/Yeast plasmid prep

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Contents

Overview

Designed to prep plasmids from yeast. Generally used to recover plasmids after screening a library. Produces low concentration DNA, suitable for PCR or transformation into E. coli but not direct sequencing. We tried using the Zymo yeast prep kit and had similar results (enough DNA to transform, but not to sequence).

Procedure

Materials

  • SDS/NaOH. Make fresh each time.
    • 10mM Tris, pH 7.5
    • 1mM EDTA, pH 8.0
    • 3% SDS
    • 200mM NaOH
  • TE/NaOAc
    • 10mM Tris, pH 7.5
    • 1mM EDTA, pH 8.0
    • 600mM NaOAc, pH 5.2
  • Phenol/Chloroform
  • 100% Ethanol
  • 3M Sodium Acetate, pH 5.2

Method

  • Grow an overnight culture in appropriate media
  • Pellet 1.5mL cells (5 minutes, 6000g works fine)
  • Resuspend in 100uL SDS/NaOH
  • Leave at RT for 15 minutes with occasional agitation
  • Add 100uL TE/NaOAc, mix well
  • Add 200uL phenol/chloroform, vortex, spin 5 minutes at max speed
  • Remove supernatant, add 2x volume 100% EtOH, 1/10th volume 3M NaOAc
  • Leave at -20C for >30 minutes
  • Spin >10 minutes at 4C, max speed
  • Remove supernatant, wash with 70% EtOH, respin
  • Remove supernatant, air dry
  • Resuspend in 20uL buffer or water
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