Rapid isolation of yeast total DNA - MetabolicEngineeringGroupCBMA/MetabolicEngineeringGroupCBMA.github.io GitHub Wiki
This protocol is similar to Extraction of chromosomal DNA with PCA (phenol:chloroform:isoamylalcohol) one, but incorporates an ethanol precipitation at the end. This has been extensively used and usually works very well. At one point, PCR was not possible using the undiluted DNA as template, but PCR products appeared from x10 dilutions. Possibly due to oxidized phenol.
Hoffman, Charles S. 2001. “Preparation of Yeast DNA.” In Current Protocols in Molecular Biology. John Wiley & Sons, Inc.
The DNA preparation described in the Basic Protocol can be easily scaled up to prepare chromosomal DNA for use in Southern hybridization analysis (UNIT 2.9A), in vitro DNA amplification by PCR (UNIT 15.1), or Digestion and ligation (UNITS 3.1 & 3.16) to clone integrated plasmids. This procedure is significantly faster than other protocols used to isolate high-molecular-weight DNA. Although the DNA is subject to shearing in this protocol, the resulting DNA is of sufficiently high molecular weight to enable detection of 19-kb Digestion products by Southern hybridization analysis.
Additional Materials (also see Basic Protocol)
TE buffer (APPENDIX 2) 1 mg/ml DNase-free RNase A (UNIT 5.5) 4 M ammonium acetate solution (APPENDIX 2) 100% ethanol
15 ml FALCON tubes Tabletop centrifuge
1.Grow a 10-ml culture of yeast in YPD overnight to stationary phase in 50 ml sterile FALCON polypropylene tubes (see Basic Protocol, step 1).
2.Spin culture 1 min in 5000 rpm in eppendorf centrifuge, room temperature or chilled does not matter. Pour off supernatant, and resuspend cells in 0.5 ml water.
3.Transfer the resuspended cells to an eppendorf microcentrifuge tube and spin 5 sec at room temperature. Pour off supernatant. This wash step in water removes any remaining medium.
4.Disrupt pellet by vortexing briefly.
4.Resuspend cells in 200 ul Breaking Buffer. Add 0.3 g glass beads (~200 ul volume) Add 200 ul phenol/chloroform and vortex at highest speed for 3 min.
The phenol/chloroform is the LOWER phase in the stock container, the upper phase is buffer. The amount of vortexing required can vary depending upon the vortex used. Determine by microscopic examination the minimum vortexing required for a particular machine to break 80% to 90% of the cells. If using a multi-tube vortex rather than a single-tube vortex, the time of vortexing may need to be increased up to 4 or 5 min to get more efficient breakage of cells. However, longer vortexing can result in shearing of DNA molecules.
5.Add 200 ul TE buffer and vortex briefly.
6.Microcentrifuge 5 min at high speed, room temperature, and transfer aqueous layer to a clean microcentrifuge tube. Add 1 ml of 99% ethanol and mix by inversion.
7.Microcentrifuge 3 min at high speed, room temperature.
8.Remove supernatant and resuspend pellet in 400uL TE buffer.
8.Add 30 ul of 1 mg/ml or 1.5 uL of a 20 mg/ml solution (0.03mg) RNase A (free from DNase), mix, and incubate 5 min at 37C.
9.Add 10 ul of 4 M ammonium acetate (or 4 uL of 10M) and 1 ml of 100% ethanol. Mix by inversion.
10.Microcentrifuge 3 min at high speed, room temperature. Discard supernatant and dry pellet.
11.Resuspend DNA in 100 - 200 ul TE buffer. Yields of ~20 ug of chromosomal DNA should be obtained. This DNA is ready to use for ++Digestion_ (UNIT 3.1), in vitro PCR amplification (UNIT 15.1), or Southern blot analysis (UNIT 2.9). For Southern blots, best results are obtained when 5 ul DNA (~1 ug) is digested in a total volume of 20 ul. To amplify by PCR, 2 ul of DNA should be used in a 50 ul reaction.
Breaking Buffer 2% (v/v) Triton X-100 1% (v/v) sodium dodecyl sulfate (SDS) 100 mM NaCl 10 mM Tris⋅Cl, pH 8.0 1 mM EDTA, pH 8.0 Store ≤1 year at room temperature