Genomic DNA Prep (Bust ‘n’ Grab Protocol)-PDF

Overview

This protocol is for rapidly isolating genomic DNA from an overnight culture of yeast. This protocol was adapted from Harju, et al (2004). Please see the reference below and cite it if you use this protocol.

Materials

• Overnight yeast culture (grown for 20-24h) in YPD
• Dry ice-ethanol bath
• Lysis buffer (2% Triton X-100, 1% SDS, 100mM NaCl, 10mM Tris-HCL, pH 8.0, 1mM EDTA, pH 8.0)
• Chloroform
• 100% Ethanol (ice-cold, put in -20°C before you start this protocol)
• 70% Ethanol
• TE (10mM Tris, pH 8.0, 1mM EDTA, pH 8.0)
• (optional) RNase cocktail (Ambion)

Protocol

1. Transfer 1.5 ml of liquid culture of yeast grown for 20 – 24 h at 30°C in YPD into a microcentrifuge tube. Pellet cells by centrifugation at 20,000 × g for 5 minutes.
2. Add 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).
3. Immerse tubes in a dry ice-ethanol bath for 2 minutes, transfer to in a 95°C water bath for 1 minute. Repeat; vortex 30 seconds.
4. Add 200 μl of chloroform; vortex 2 minutes.
5. Centrifuge 3 minutes, room temperature, 20,000 × g.
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, 5 minutes. Alternatively, precipitate at -20°C to increase yield.
8. Centrifuge 5 minutes, room temperature, 20,000 × g. Remove supernatant with a pulled Pasteur pipette by vacuum aspiration.
9. Wash the pellet with 0.5 ml 70% ethanol, spin down as described in step 8 above. Remove supernatant.
10. Air-dry the pellets at room temperature or for 5 minutes at 60°C in a vacuum dryer.
11. Resuspend in 25–50 μl TE [10 mM Tris (pH 8.0), 1 mM EDTA (pH 8.0)] or water. Samples obtained directly from plates should be resuspended in a 10 μl volume, because the yield will be smaller. 0.25 μl RNase cocktail (Ambion) should be added to the samples used for Southern blot hybridization (final concentration 0.125 U RNAse A, 5 U RNase T1).

References

• Harju, S and H. Fedosyuk and KR Peterson (2004) Rapid isolation of yeast genomic DNA: Bust n’ Grab BMC Biotechnol 4:8