Yeast DNA Prep: Difference between revisions
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#resuspend pellet in 200 ul breaking buffer | #resuspend pellet in 200 ul breaking buffer | ||
#wear gloves and add: | #wear gloves and add: | ||
#*200 | #*200 ul phenol:choloroform:isoamyl alcohol (25:24:1) | ||
#*200 | #*200 ul (@200 mg) glass beads | ||
#close cap tightly and vortex for 2.5 min. | #close cap tightly and vortex for 2.5 min. | ||
#*Be careful when vortexing; label can be dissolved by the phenol. | #*Be careful when vortexing; label can be dissolved by the phenol. | ||
#*Hold cap tightly so it doesn't open or spill. | #*Hold cap tightly so it doesn't open or spill. | ||
#add 200 | #add 200 ul TE buffer and spin for 5 min, in microfuge | ||
#transfer 350 ml aqueous (top) layer to fresh eppendorf. | #transfer 350 ml aqueous (top) layer to fresh eppendorf. | ||
#add 1 ml 95% ethanol and mix well, let sit for 10 minutes | #add 1 ml 95% ethanol and mix well, let sit for 10 minutes | ||
#spin for 2 min, take off supernatant, and let dry upside down 10 min. | #spin for 2 min, take off supernatant, and let dry upside down 10 min. | ||
#resuspend pellet in 50 | #resuspend pellet in 50 ul TE buffer or water. | ||
You can now use 1-2 ul of this crude yeast plasmid DNA prep to transform E. coli. | You can now use 1-2 ul of this crude yeast plasmid DNA prep to transform E. coli. |
Revision as of 11:08, 5 March 2006
This protocol is used for recovering plasmids from yeast cultures.
Protocol
- grow up yeast culture to appropriate density (near saturation)
- spin 1.5 mls of culture for 1 min in microfuge and aspirate off supernatant
- resuspend pellet in 200 ul breaking buffer
- wear gloves and add:
- 200 ul phenol:choloroform:isoamyl alcohol (25:24:1)
- 200 ul (@200 mg) glass beads
- close cap tightly and vortex for 2.5 min.
- Be careful when vortexing; label can be dissolved by the phenol.
- Hold cap tightly so it doesn't open or spill.
- add 200 ul TE buffer and spin for 5 min, in microfuge
- transfer 350 ml aqueous (top) layer to fresh eppendorf.
- add 1 ml 95% ethanol and mix well, let sit for 10 minutes
- spin for 2 min, take off supernatant, and let dry upside down 10 min.
- resuspend pellet in 50 ul TE buffer or water.
You can now use 1-2 ul of this crude yeast plasmid DNA prep to transform E. coli.
Materials
- breaking buffer
- 2% (v/v) Triton X-100
- 1% (w/v) SDS
- 100 mM NaCl
- 10 mM Tris-Cl, pH 8.0
- 1 mM EDTA, pH 8.0
- T.E. buffer (pH 8.0)
- 10 mM Tris-Cl, pH 8.0
- 1 mM EDTA, pH 8.0
- chilled phenol:choloroform:isoamyl alcohol (25:24:1)
- chilled 95% ethanol
- acid-washed glass beads (Sigma, G 3753, See CPMB, 13.12.1)