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. | ||
| Line 34: | Line 34: | ||
From http://stausta.web.wesleyan.edu/mbb294/Expt3.html | From http://stausta.web.wesleyan.edu/mbb294/Expt3.html | ||
==BioCoder version== | |||
Following is the Yeast DNA Prep protocol in BioCoder, a high-level programming language for expressing biology protocols. What you see here is the auto-generated text ouput of the protocol that was coded up in BioCoder (see Source code). More information about BioCoder can be found on my home page. Feel free to mail me your comments/ suggestions.[[User:Vaishnavi Ananth|Vaishnavi]] | |||
====Text Output==== | |||
[[Yeast DNA Prep protocol]] | |||
====Source Code==== | |||
[[Yeast DNA Prep protocol - source code]] | |||
[[Category:Protocol]] | |||
[[Category:In vitro]] | |||
[[Category:Yeast]] | |||
[[Category:DNA]] | |||
Latest revision as of 02:41, 19 November 2009
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)
From http://stausta.web.wesleyan.edu/mbb294/Expt3.html
BioCoder version
Following is the Yeast DNA Prep protocol in BioCoder, a high-level programming language for expressing biology protocols. What you see here is the auto-generated text ouput of the protocol that was coded up in BioCoder (see Source code). More information about BioCoder can be found on my home page. Feel free to mail me your comments/ suggestions.Vaishnavi
