Drummond:Yeast Genomic DNA Prep Protocol: Difference between revisions
(New page: Yeast Genomic DNA Prep Protocol 1) Inoculate a 3 mL YPD culture with a single yeast colony and grow to saturation (overnight) at 30C with shaking or rotation. 2) Transfer 500 uL or 750...) |
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Yeast Genomic DNA Prep Protocol | == Yeast Genomic DNA Prep Protocol == | ||
1) Inoculate a 3 mL YPD culture with a single yeast colony and grow to saturation (overnight) at 30C with shaking or rotation. | 1) Inoculate a 3 mL YPD culture with a single yeast colony and grow to saturation (overnight) at 30C with shaking or rotation. | ||
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3) Remove wash using a vacuum trap being careful to not disturb the pellet. | 3) Remove wash using a vacuum trap being careful to not disturb the pellet. | ||
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4) Add 0.2ml DNA Extraction Buffer | 4) Add 0.2ml DNA Extraction Buffer | ||
0.2ml phenol:chloroform:isoamyl alcohol (25:24:21) | 0.2ml phenol:chloroform:isoamyl alcohol (25:24:21) | ||
0.3g glass beads (use tube measurement) | 0.3g glass beads (use tube measurement)</nowiki> | ||
Revision as of 11:59, 25 November 2008
Yeast Genomic DNA Prep Protocol
1) Inoculate a 3 mL YPD culture with a single yeast colony and grow to saturation (overnight) at 30C with shaking or rotation.
2) Transfer 500 uL or 750 uL of cells to a microcentrifuge tube and pellet at top speed for 10 sec. Dump supernatant and wash cells with 0.5ml distilled water, and pellet at top speed for 10 sec.
3) Remove wash using a vacuum trap being careful to not disturb the pellet.
4) Add 0.2ml DNA Extraction Buffer 0.2ml phenol:chloroform:isoamyl alcohol (25:24:21) 0.3g glass beads (use tube measurement)
5) Vortex for 3 min. Add 0.2ml TE (pH 8), and transfer entire contents to a phase-lock tube.
6) Centrifuge for 5 min at top speed, and transfer the aqueous top phase to a clean microcentrifuge tube.
7) Add 1ml of 100% EtOH. Mix by inversion.
8) Centrifuge for 2 min at top speed. Remove supernatant using a vacuum trap being careful to not disturb the pellet.
9) Dissolve pellet in: 0.4ml of TE.
10) Add 5µl of 10mg/ml RNase A and mix by inversion. Incubate for 30 min at 37°C in water bath. Add 10µl of 4M ammonium acetate and 1ml of 100% EtOH. Mix by inversion.
11) Centrifuge for 2 min. Remove supernatant using a vacuum trap being careful to not disturb the pellet.
12) Air dry pellet or dry in vacuum oven for 10 min and resuspend in 50µl of EB. Check concentration with the Nanodrop spectrophotometer.
13) Optional: check the quality of the genomic DNA prep by electrophoresis on a 0.8% agarose gel.
