10.2.1 genomic DNA extraction

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10.2.1 Genomic DNA extraction (embryo)

Hiroyuki Takeda
University of Tokyo

http://medaka.lab.nig.ac.jp/

10.2.1.1 Genomic DNA extraction

1. Wash each embryo with DDW and put each embryo into 1.5 ml tube.
2. Freeze embryos at -80C
3. Add 200 ul of TNES-6U buffer (10mM Tris-HCl, pH 7.5; 125mM Na Cl; 10mM EDTA; 1% SDS; 6M Urea) and homogenize with a plastic pestle.
4. Add 5 ul of protenase K solution (20 mg/ml) and incubate at 37 C over night.
5. Add 300 ul of phenol/chloroform solution (TE saturated phenol : chloroform : Isoamyl Alcohol = 25 : 24 : 1) and mix well for 10 minutes by inversion.
6. Centrifuge at 10,000 rpm at room temperature.
7. Transfer aqueous phase to a new tube and add 500 ul of diethyl ether, mix by inversion.
8. Centrifuge at 5,000 rpm for 2 minutes and remove ether and lipid phase.
9. Add 20ul of 5M Na Cl and 500ul of 100% cold Ethanol (-20 C) to the aqueous phase and incubate at -80C for 1 hour.
10. Centrifuge at 15,000 rpm for 15 minutes at 4C.
11. Remove supernatant and add 1000ul of cold 70% Ethanol (-20 C).
12. Centrifuge at 15,000rpm for 10 minutes at 4C and remove supernatant.
13. After drying add 250 ul of TE solution.