[問題] 請問小鼠尾巴genomic DNA萃取,DNA沉澱步驟相關問題

看板Biotech (生命科學)作者oopa (11)時間18年前 (2007/10/09 19:41)推噓5(5推 0噓 5→)

留言10則, 5人參與討論串1/1

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. -- ※ 發信站: 批踢踢實業坊(ptt.cc) ◆ From: 211.76.175.170