血液DNA的提取
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Part A: Purifying nuclear pellets.
1) Add 50-60μl fresh, packed red blood cells (RBC) to 700μl PBS. Mix by inversion.
2) Add 700-800μl Lysis buffer. Close tube. Vortex briefly (2-5s)
3) Centrifuge for 15s at 12,000 rpm. Remove red supernatant by aspiration.
4) Add 1.0ml new Lysis buffer. Vortex (15-30s) to break up pellet.
6) Centrifuge for 15s at 12,000 rpm. Remove red supernatant by aspiration.
Part B: Extraction of DNA from nuclei.
1) Transfer pellets to 50ml tubes to digest by either:
a) Adding 1ml TEN buffer to pellet in microfuge tube. Vortex well (>30s) to break up pellet. Pour contents into empty 50ml tube. Add 5ml TEN w/ 0.5%SDS. OR
b) Use loop to move pellet into empty 50 ml tube. Add 6ml TEN w/ 0.5%SDS.
2) Add 120μl proteinase-K. Swirl to mix.
3) Incubate tubes at 55℃ for 12 hrs to 3 days with occasional vigorous mixing.
11) Hook out large clots with plastic loop and transfer to microfuge tube pre-filled with 1.0ml 70-80% Ethanol. Let sit at 4℃ for 20min to 2 days.
12) Spin pellets at 12,000 rpm for 3-5min. Remove Ethanol wash by aspiration.
13) Let pellet dry 5-30min at RT. Add 200-500μl TE soln and let resuspend at 4℃.
14) Check DNA concentration on flourometer and quality on gel. Adjust as needed.
