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。
, j4 J! Y! D6 s$ y. YEthanol Precipitation of DNA& {: k6 f) m6 D
This procedure allows the concentration of DNA samples from dilute solution and the removal of unwanted salts from DNA samples.
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3 E9 {; n0 F3 d; b4 B2 l6 u•3M Sodium Acetate buffer, pH 5.2, M( y+ p2 D, ~8 W# L4 [- @" q
•Cold 100% Ethanol
5 o3 u: z* O/ l5 Y•Cold 70% Ethanol in sterile dH2O % L, @6 `( F. D0 g( V: e0 |$ E; J% T
•DNA sample6 U  R' e( X* p; H1 L* q) `! J2 F
•4 ℃ Microcentrifuge
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Procedure
7 W3 ~8 r: t5 s# }% {2 Q; t; ^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).1 r$ U2 U/ |. @! d
2.Add one tenth volume of Sodium Acetate buffer to equalize ion concentrations.
3 E" ]1 v% J  D, q8 B/ w7 S3.Add at least two volumes of cold 100% ethanol; let stand in -20℃ freezer for at least one hour .
2 J& `3 g) J  z3 B4.Centrifuge sample for 15 minutes at highest speed in a 4℃ microcentrifuge .
. S9 u/ D- ~" b8 j" L6 R5.Remove as much supernatant as possible with a 1 mL micropipet; recentrifuge, then remove the rest with a 200 μl pipet.
1 S7 N0 m, w- H1 y% f6.Add 200 μl of cold 70% ethanol; centrifuge for 5 minutes in a 4 ℃ centrifuge.; E4 B4 T6 x8 \+ g
7.Remove supernatant with a 200 μl pipet; keep for 5 minites at RT. ! E! o/ c" z3 h1 h
8.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。