以往用傳統方法(phenol-chloroform)萃取小鼠尾的genomic DNA 所得之產物將用於genotyping(PCR) 以往於"以absolute EtOH沉澱DNA"這個步驟中 只要加入兩倍sample體積的EtOH invert幾次就可以看到漂亮的棉絮狀DNA沉澱出來 但是隔了約兩個月再做 卻發現一加ETOH就變成全部混濁 完全不覆以往的棉絮狀沉澱 非常困擾 希望有好心人提供看法跟建議 什麼都可以 感謝! Lysis buffer: NaCl 100 mM Tris (pH8) 50 mM EDTA (pH8) 50 mM SDS 1% Proteinase K 200 ug/ml (freshly prepared and added) 步驟 轫 1. Freshly prepare lysis buffer, add Proteinase K (Stock 2 mg/ml, 1ml/tube; stored at -30℃) to final concentration=200 ug/ml (10x dilution). 轫 2. Add 500 ul (can be modified to 700 ul) lysis buffer to mouse tail sample. 顷 55℃, gently shaking O/N (12~16hr) 轫 3. Add 500 ul (or 1V lysis buffer) phenol, invert 5-10 times. 顷 12000 rpm, 5 min, RT 轫 4. Collect upper water phase solution into new microtube, Add 500 ul (1V) phenol chloroform IAA (24:25:1), invert 5-10 times. 顷 12000 rpm, 5 min, RT 轫 5. Collect upper water phase solution into new microtube, Add 500 ul (1V) chloroform, invert 5-10 times. 顷 12000 rpm, 5 min, RT 轫 6. Collect upper water phase solution into new microtube, add 900 ul 100% ethanol invert upside down until transparent or white precipitate appears. 轫 7. Pellet down the precipitate, discard supernatant. Add 70% ethanol. 顷 12000 rpm, 3 min, RT 轫 8. Discard supernatant (suction out the residual liquid drops, if necessary) air dry to vaporize the ethanol. 轫 9. Add 42℃ ddH2O according to desired concentration, stand for complete dissolving DNA. (Gently tapping the tube helps disslving) 轫 10. Store at 4℃ temporally. After experiment, transfer DNA to 4℃ for long term storage. --

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