Quint Lab:yeast plasmid prep
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Revision as of 04:38, 17 October 2008 by
Marcel Quint
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yeast plasmid prep
inoculate 2 ml of Glucose medium and let grow over night @ 30°C
spin down 1.5 ml of the over night culture and resuspend in0.5 ml S-buffer
incubate 30 min, 37°C
add 0.1 ml lysis solution, vortex
incubate 30 min, 65°C
add 0.166 ml potassium acetate and mix, chill on ice for 10 min
spin down for 10 min, pour the SN into a new tube (2 ml!)
DNA precipitation: add 0.8 ml EtOH, 10 min on ice, spin down for 10 min, pour off the SN
wash DNA with 0.5 ml 70% EtOH, dry pellet
dissolve DNA in 0.04 ml water
S-buffer:
10 mM K2HPO4; pH 7.2
10 mM EDTA
50 mM 2-Mercaptoethanol
50 µg/ml zymolase
lysis solution:
25 mM Tris-HCl; pH 7.5
25 mM EDTA
2.5% SDS
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