乙醇沉淀DNA for new guys

chochochocho

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最近好多小童鞋困扰于酶切连接问题，涉及到一些分子实验基本操作，会陆陆续续发一些实用的Protocol，欢迎大家一块儿来modify！希望对新手们有帮助。
7 P6 _' t8 L" F; QEthanol Precipitation of DNA9 n# b' s) B  \3 r' H4 x) y; O
This procedure allows the concentration of DNA samples from dilute solution and the removal of unwanted salts from DNA samples.
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•3M Sodium Acetate buffer, pH 5.2% ~- G% F6 |: o/ T
•Cold 100% Ethanol 1 m  `9 N4 c, e- {* B8 f  @
•Cold 70% Ethanol in sterile dH2O   q( Q1 `4 Y" e+ ~
•DNA sample
  N: c* S& a8 W5 a0 S3 `•4 ℃ Microcentrifuge 2 ?: c: K' I8 V6 |, O

5 ^- V6 C% H* _+ ]( j0 r1 w0 O8 WProcedure2 y' K7 v, y7 a
1.Transfer DNA to a container where it fills one fourth the total volume (a 500 μl tube should have no more than 125 μl of DNA solution, for example)." ~" Q; ]7 U& C& t# g" h
2.Add one tenth volume of Sodium Acetate buffer to equalize ion concentrations.& ~' ~7 h# a8 i: L# z1 j: }/ Z1 U: m
3.Add at least two volumes of cold 100% ethanol; let stand in -20℃ freezer for at least one hour .
* j8 D! B7 e1 p+ X/ c# ]4.Centrifuge sample for 15 minutes at highest speed in a 4℃ microcentrifuge .
4 A" U1 q9 q, _$ ?' N  q% A9 {2 q/ Q2 A5.Remove as much supernatant as possible with a 1 mL micropipet; recentrifuge, then remove the rest with a 200 μl pipet.. S) s) R1 n- K! \  n+ M/ \" s
6.Add 200 μl of cold 70% ethanol; centrifuge for 5 minutes in a 4 ℃ centrifuge.7 j2 d8 x; J' c# j
7.Remove supernatant with a 200 μl pipet; keep for 5 minites at RT.
( C/ ]: J. W5 F6 c8.Resuspend pellet in desired volume of water or TE buffer.

老虎菜998

酶切是否成功主要决定于DNA的质量。而通过乙醇沉淀得到的DNA质量变异较大，纯化方法、操作习惯、干燥时间、溶解体积等等都可能成为最终试验失败的原因。如果条件许可的话，本人还是建议采用离心柱的方法纯化DNA，该法尽管纯度不是很高，但用于酶切还是可以的，关键中影响因素较小，重复性好，对于新手来说出错的可能也小。