Sep 06, 2022
DNA extraction protocol
- Cecile Grondin1,
- Jean-Luc Legras1,
- hugo.devillers2
- 1CIRM-Levures, SPO, INRAE, France;
- 2ADEL, SPO, INRAE, France
Protocol Citation: Cecile Grondin, Jean-Luc Legras, hugo.devillers 2022. DNA extraction protocol . protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvorx47v4o/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: September 05, 2022
Last Modified: September 06, 2022
Protocol Integer ID: 69564
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Abstract
This DNA extraction protocol has been developed at the CIRM-Levures (INRAE, SPO, FRANCE) to extract yeast chromosomes. It is based on a phenol/chloroform DNA extraction procedure.
CELL LYSIS AND NUCLEIC ACID EXTRACTION
CELL LYSIS AND NUCLEIC ACID EXTRACTION
- Perform a 3 ml YPD (yeast extract 10g/l, bacto peptone 10g/l, glucose 10g/l) culture for 60h in order to harvest cells at stationary phase.
- Centrifuge 5 min at 5000 rpm and remove the supernatant.
- Wash the pellet with 5 ml of 50mM EDTA, transfer to a 2 ml screw cap tube and centrifuge for 5 min at 12000 rpm. Remove the supernatant.
- Add 0.2ml lysis buffer (50mM Tris pH 8, 50mM EDTA, 100mM NaCl, 2% Triton, 1% SDS), 0.2ml TE 1X (10mM Tris pH 8, 1mM EDTA), 0.2ml phenol/chloroform and 0.3g glass beads.
- Grind with the Digital Disruptor Genie (Scientific Industries, Bohemia, NY 11716 U.S.A) for 2 minutes at 2850 rpm.
- Centrifuge 5 min at 12000 rpm.
- Transfer the aqueous phase to a new tube, add 0.5 ml of chloroform and mix.
- Centrifuge 5 min at 12000 rpm.
- Transfer the aqueous phase to a new tube, add 0.5 ml of isopropanol and mix.
- Centrifuge for 5 min at 12000 rpm.
- Wash the pellet with 0.4 ml of 70% ethanol and let the tube dry.
- Re-suspend the DNA pellet in 490 µL of TE 1X and let overnight at 4 °C.
- Add 10 μl of RNase (100 mg/ml, Qiagen ref: 19101). Incubate for 60 min at 37 °C.
DNA PURIFICATION, Magnetic Beads BINDING
DNA PURIFICATION, Magnetic Beads BINDING
- To each sample, add 50 μl of 5 M NaCl, 15 μl of Chemagic CMG-252-A magnetic bead (PerkinElmer, Waltham, Massachusetts, U.S.A) suspension, 250 μl of 7.8M Guanidium chloride and 800 μl of isopropanol.
- Mix by inversion for 5 minutes.
- Set up the tubes on the magnetic rack (DynaMag™-2 Magnet type, Invitrogen ThermoFisher Scientific).
- Wait for 2 min. Remove the liquid.
- Perform a first wash with 1 ml of AMMLAV/E buffer (10 mM Tris pH 8.0, 0.1 mM EDTA, 60 mM potassium acetate, 65% ethanol) and mix gently to disintegrate the beads.
- Place the tubes on the magnetic rack. Wait 2 min. Remove the liquid.
- Repeat a second wash with 1 ml of AMMLAV/E buffer.
- Perform two more washes as above with 1 ml of 75% ethanol
- Air dry the beads at room temperature for 5 min.
- Add 0.1 ml of 1X TE. Mix well to re-suspend the beads in TE 1X. Incubate 10 min at room temperature.
- Place the tubes on the magnetic rack. Wait 5 min. Transfer the DNA solution to new storage tubes.
- The DNAs are then stored at -20°C.