作者oopa (11)
看板Biotech
标题[问题] 请问小鼠尾巴genomic DNA萃取,DNA沉淀步骤相关问题
时间Tue Oct 9 19:41:58 2007
以往用传统方法(phenol-chloroform)萃取小鼠尾的genomic DNA
所得之产物将用於genotyping(PCR)
以往於"以absolute EtOH沉淀DNA"这个步骤中
只要加入两倍sample体积的EtOH
invert几次就可以看到漂亮的棉絮状DNA沉淀出来
但是隔了约两个月再做
却发现一加ETOH就变成全部混浊
完全不覆以往的棉絮状沉淀
非常困扰
希望有好心人提供看法跟建议
什麽都可以 感谢!
Lysis buffer:
NaCl 100 mM
Tris (pH8) 50 mM
EDTA (pH8) 50 mM
SDS 1%
Proteinase K 200 ug/ml (freshly prepared and added)
步骤
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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1F：推 daniel2806:应该是isoporpanol吧 10/10 00:00
2F：推 huangsw:我们家也是用绝对酒精 10/10 00:06
3F：推 chvkimo:我听过 isopropanol 效果比酒精好 (酒精也可以用) 10/10 13:59
4F：→ chvkimo:有人可以分享一下 或做精确的解说吗? 10/10 14:00
5F：推 oopa:问题是以前用绝对酒精效果很好.现在却不行,难道酒精吸水了?ꐠ 10/10 22:10
6F：推 pianostar:isopropanol 沉淀效果好 但是盐类残留多 洗盐要多洗几次 11/04 04:41
7F：→ pianostar:酒精沉淀 盐类残留少 沉淀效果没异丙醇好~ 11/04 04:43
8F：→ pianostar:纯酒精的确会吸水 所以 存放的无水酒精要用parafilm封口 11/04 04:44
9F：→ pianostar:根据本人的经验 沉淀genomic DNA 如果雾雾一片 11/04 04:45
10F：→ pianostar:DNA应该是断光光的状态喔!!@@ 11/04 04:45