Isolation of DNA from yeast cells


  • collect cells of 1,5 ml of a saturated culture
  • decant supernatant and suspend cells in the remaining drop (about 100 µl)
  • add 200 µl of extraction buffer
  • add 200 µl of phenol/chloroform/iA
  • add glass beads (ca 0,3 g)
  • thoroughly vortex for 2 min
  • spin 5 min, 13k
  • transfer the aqueous phase to a new tube, add 500 µl Ethanol
  • mix and spin 10min, RT
  • wash pellet with 70% ethanol, dry with speed vac (1 min)
  • dissolve pellet in 50 µl TE

Isolation of plasmid

transform electrocompetent E. coli with 1 µl of the DNA prep
select for AmpR
using e. g. E. coli KC8, the auxotrophic yeast markers can be used to complement mutations of KC8, e.g trpC9830 is complemented by TRP1, AmpR, Trp+ transformants are selected on minimal medium

materials:

extraction buffer:
2% tritonX-100
1% SDS
100 mM NaCl
10 mM Tris/HCl pH8
1 mM EDTA

phenol/chloroform
Phenol (sat. TE, pH 8): Chloroform: i-Amylalkohol= 25:24:1

glass beads:
0,45-0,5 mm, B. Braun Melsungen AG, autoklaved