Drummond:Yeast Genomic DNA Prep Protocol: Difference between revisions
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* Inoculate a 3 mL YPD culture with a single yeast colony and grow to saturation (overnight) at 30C with shaking or rotation. | * Inoculate a 3 mL YPD culture with a single yeast colony and grow to saturation (overnight) at 30C with shaking or rotation. | ||
* 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. | * 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. | ||
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* Remove wash using a vacuum trap being careful to not disturb the pellet. | * Remove wash using a vacuum trap being careful to not disturb the pellet. | ||
* 4)Add 0.2ml DNA Extraction Buffer | * 4)Add the following: | ||
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) | ||
* Vortex for 3 min. Add 0.2ml TE (pH 8), and transfer entire contents to a phase-lock tube. | * Vortex for 3 min. Add 0.2ml TE (pH 8), and transfer entire contents to a phase-lock tube. | ||
* Centrifuge for 5 min at top speed, and transfer the aqueous top phase to a clean microcentrifuge tube. | * Centrifuge for 5 min at top speed, and transfer the aqueous top phase to a clean microcentrifuge tube. | ||
* Add 1ml of 100% EtOH. Mix by inversion. | * Add 1ml of 100% EtOH. Mix by inversion. | ||
* Centrifuge for 2 min at top speed. Remove supernatant using a vacuum trap being careful to not disturb the pellet. | * Centrifuge for 2 min at top speed. Remove supernatant using a vacuum trap being careful to not disturb the pellet. | ||
* Dissolve pellet in: 0.4ml of TE. | * Dissolve pellet in: 0.4ml of TE. | ||
* 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. | * 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. | ||
* Centrifuge for 2 min. Remove supernatant using a vacuum trap being careful to not disturb the pellet. | * Centrifuge for 2 min. Remove supernatant using a vacuum trap being careful to not disturb the pellet. | ||
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* Optional: check the quality of the genomic DNA prep by electrophoresis on a 0.8% agarose gel. | * Optional: check the quality of the genomic DNA prep by electrophoresis on a 0.8% agarose gel. | ||
{{Drummond_Bottom}} | {{Drummond_Bottom}} | ||
Revision as of 12:05, 25 November 2008
Protocol
- Inoculate a 3 mL YPD culture with a single yeast colony and grow to saturation (overnight) at 30C with shaking or rotation.
- 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.
- Remove wash using a vacuum trap being careful to not disturb the pellet.
- 4)Add the following:
0.2ml DNA Extraction Buffer 0.2ml phenol:chloroform:isoamyl alcohol (25:24:21) 0.3g glass beads (use tube measurement)
- Vortex for 3 min. Add 0.2ml TE (pH 8), and transfer entire contents to a phase-lock tube.
- Centrifuge for 5 min at top speed, and transfer the aqueous top phase to a clean microcentrifuge tube.
- Add 1ml of 100% EtOH. Mix by inversion.
- Centrifuge for 2 min at top speed. Remove supernatant using a vacuum trap being careful to not disturb the pellet.
- Dissolve pellet in: 0.4ml of TE.
- 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.
- Centrifuge for 2 min. Remove supernatant using a vacuum trap being careful to not disturb the pellet.
- Air dry pellet or dry in vacuum oven for 10 min and resuspend in 50µl of EB. Check concentration with the Nanodrop spectrophotometer.
- Optional: check the quality of the genomic DNA prep by electrophoresis on a 0.8% agarose gel.
