Drummond:DNA Prep

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
m (Yeast Genomic DNA Prep)
(bust 'n' grab)
Line 4: Line 4:
== Yeast Genomic DNA Prep ==
== Yeast Genomic DNA Prep ==
-
* 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 1.5 mL of cells to a microcentrifuge tube 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.
+
# 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).
-
 
+
# 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.
-
* Remove wash using a vacuum trap being careful to not disturb the pellet.
+
# Centrifuge for 3 minutes at 20,000g 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.
-
* Add the following: <br>    0.2ml DNA Extraction Buffer <br>    0.2ml phenol:chloroform:isoamyl alcohol (25:24:21) <br>    0.3g glass beads (about 400 uL)
+
# Incubate at room temperature for 5min. Alternatively, precipitate at -20°C to increase yield.
-
 
+
# Centrifuge 5min at 20,000g at room temperature. Aspirate supernatant using a vacuum trap.
-
* Vortex for 3 min.  Add 0.2ml TE (pH 8), and transfer entire contents to a phase-lock tube.  
+
# 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.
-
* Centrifuge for 5 min at top speed, and transfer the aqueous top phase to a clean microcentrifuge tube.
+
# Resuspend in 25-50μL water or TE.
-
 
+
-
* Add 1ml of 100% EtOH. Mix by inversion.  
+
-
 
+
-
* Centrifuge for 2 min at top speed. Remove supernatant using a vacuum trap being careful not to 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.
+

Revision as of 11:20, 14 August 2012

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-1000, 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. Centrifuge for 3 minutes at 20,000g at room temperature.
  6. Transfer the upper aqueous phase to a microcentrifuge tube containing 400 μL ice-cold 100% ethanol. Mix by inversion or gentle vortexing.
  7. Incubate at room temperature for 5min. Alternatively, precipitate at -20°C to increase yield.
  8. Centrifuge 5min at 20,000g at room temperature. Aspirate supernatant using a vacuum trap.
  9. Wash the pellet with 0.5 mL 70% ethanol, spin down as described in step 8 above. Remove and discard supernatant.
  10. Air-dry the pellets at room temperature or for 5min at 60°C in a vacuum dryer.
  11. Resuspend in 25-50μL water or TE.
Personal tools