Drummond:DNA Prep

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(bust 'n' grab)
Current revision (10:44, 13 October 2012) (view source)
(Chloroform step added)
 
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== Yeast Genomic DNA Prep ==
== Yeast Genomic DNA Prep ==
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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.  
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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.
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# Transfer 1.5 mL of cells to a microcentrifuge tube and pellet at top speed for 10 sec.
+
# Transfer 1.5 mL of cells to a microcentrifuge tube and pellet at top speed for 10 sec.
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# Resuspend pellet in 200 μL of lysis buffer (2% Triton X-1000, 1% SDS, 100 mM NaCl, 10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0).
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# Resuspend pellet in 200 μL of lysis buffer (2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0).
# Immerse tubes in a dry ice-ethanol bath for 2 minutes, transfer to a 95°C water bath for 1 minute. Repeat. Vortex for 30s.
# Immerse tubes in a dry ice-ethanol bath for 2 minutes, transfer to a 95°C water bath for 1 minute. Repeat. Vortex for 30s.
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# Centrifuge for 3 minutes at 20,000g at room temperature.
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# Add 200 μL of chloroform; vortex 2 minutes.
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# Centrifuge for 3 minutes at 14,000g at room temperature.
# Transfer the upper aqueous phase to a microcentrifuge tube containing 400 μL ice-cold 100% ethanol. Mix by inversion or gentle vortexing.
# Transfer the upper aqueous phase to a microcentrifuge tube containing 400 μL ice-cold 100% ethanol. Mix by inversion or gentle vortexing.
# Incubate at room temperature for 5min. Alternatively, precipitate at -20°C to increase yield.
# Incubate at room temperature for 5min. Alternatively, precipitate at -20°C to increase yield.
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# Centrifuge 5min at 20,000g at room temperature. Aspirate supernatant using a vacuum trap.
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# Centrifuge 5min at 14,000g at room temperature. Aspirate supernatant using a vacuum trap.
# Wash the pellet with 0.5 mL 70% ethanol, spin down as described in step 8 above. Remove and discard supernatant.
# Wash the pellet with 0.5 mL 70% ethanol, spin down as described in step 8 above. Remove and discard supernatant.
# Air-dry the pellets at room temperature or for 5min at 60°C in a vacuum dryer.
# Air-dry the pellets at room temperature or for 5min at 60°C in a vacuum dryer.
# Resuspend in 25-50μL water or TE.
# Resuspend in 25-50μL water or TE.

Current revision

Introduction

To confirm insertion location and sequence of genetic constructs in the yeast genome, or amplify sections of the genome for manipulation, one can either PCR using boiled cells (colony PCR) or extract genomic DNA. We find that taking 1–1.5h to prepare clean genomic DNA makes all subsequent steps easier and more reliable.

Yeast Genomic DNA Prep

  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 1.5 mL of cells to a microcentrifuge tube and pellet at top speed for 10 sec.
  3. Resuspend pellet in 200 μL of lysis buffer (2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0).
  4. Immerse tubes in a dry ice-ethanol bath for 2 minutes, transfer to a 95°C water bath for 1 minute. Repeat. Vortex for 30s.
  5. Add 200 μL of chloroform; vortex 2 minutes.
  6. Centrifuge for 3 minutes at 14,000g at room temperature.
  7. Transfer the upper aqueous phase to a microcentrifuge tube containing 400 μL ice-cold 100% ethanol. Mix by inversion or gentle vortexing.
  8. Incubate at room temperature for 5min. Alternatively, precipitate at -20°C to increase yield.
  9. Centrifuge 5min at 14,000g at room temperature. Aspirate supernatant using a vacuum trap.
  10. Wash the pellet with 0.5 mL 70% ethanol, spin down as described in step 8 above. Remove and discard supernatant.
  11. Air-dry the pellets at room temperature or for 5min at 60°C in a vacuum dryer.
  12. Resuspend in 25-50μL water or TE.
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