- 0.5 to 1 cm tail digested in 500 ul SE buffer/proteinase K, 55°C overnight.
- Add pre-warmed saturated NaCl solution(6M) 170 ul, gently mix to a final concentration of 1.5 M.
- Add 670 ul of chloroform and mix for 30 to 60 minutes by gentle rotation.
- Centrifugate at 12000 rpm for five minutes.
- Transfer the supernatant to a fresh tube, add one volume of isopropanol at room temperature and gently mix for five minutes.
- Pick DNA or centrifugate to collect DNA.
- Wash with 70 percent ethanol for one hour or overnight.
- Spin and dry.
- Dissolve in Tris 10 mM, pH8.5.
SE Buffer/Proteinase K
|
Final Concentration |
Stock Concentration |
For 10 ml Mix |
|
| NaCl | 75 mM | 5 M | 150 µl |
| EDTA pH8.0 | 25 mM | 0.5 M | 500 µl |
| SDS | 1% | 10% | 1000 µl |
| H2O | 8150 µl | ||
| Proteinase K | 200 µg/ml | 10 mg/ml | 200 µl |
Reference
R. MGllenbach, PJL Lagoda and C Welter (1989). An Efficient Salt-Chloroform Extraction of DNA from blood and tissues. Trend in Genetics 5(12):391.