Quick Genomic DNA Preparation
From CCGB
[edit]
Quick Genomic DNA Preparation
--by Hao Wang
a. 2 × Modified Bradley’s solution
Stock 20 mM Tris, pH 7.5 2 M 5 ml 4 mM EDTA 0.5 M 4 ml 20 mM NaCl 5 M 2 ml 1% SDS 10% 50 ml dd H2O 439 ml Total 500 ml
Before use, add 2 mg Prot.K/ml.
b. 75 mM NaOAc in 100% EtOH
Stock 75mM NaOAc(3 M NaOAc) 12.5 ml 100% EtOH 487.5 ml Total 500 ml
1. Pellet up to 3 × 107 cells.
2. Resuspend om PBS (200 μl). Transfer to microfuge tube.
3. Add 200 μl 2 × Bradley’s solution with Proteinase K. Incubate o/n @ 55°C.
4. Overlay with 800 μl cold 75 mM NaOAc in EtOH. Hold 20 min. Don’t vortex.
5. Mix gently. Hold 20min. Don’t vortex.
6. Mix gently. Hold 20min.
7. Vortex briefly. Spin @ 10000rpm for 5 min.
8. Remove supernatant and discard. Add 500 μl 70% EtOH. Loosen pellet from bottom. Hold for 20 min.
9. Centrifuge @ 10000 rpm for 5 min. Repeat wash, hold for 5 min. Centrifuge 10000 rpm for 5 min.
10. Allow to dry a bit.
11. Bring up to 30-40 μl TE + 0.1 μg/μl RNase. Hold @ 55°
