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。- P) K" ~: {' S7 s
Ethanol Precipitation of DNA/ C  e( U* g* C
This procedure allows the concentration of DNA samples from dilute solution and the removal of unwanted salts from DNA samples.$ Y" L2 h+ ?1 ?" o
Materials
9 w* S" N' G( }" J0 G' e•3M Sodium Acetate buffer, pH 5.2
; ^' D* O' {3 a$ n•Cold 100% Ethanol ( p: K- P" F4 p, T
•Cold 70% Ethanol in sterile dH2O ' Y8 q  L7 @9 A- p9 D8 U( R* @
•DNA sample2 |' J8 F$ J  T) H( O' c
•4 ℃ Microcentrifuge 0 f) L/ n9 j' _( n0 F4 m
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Procedure% `9 q- F2 v/ x6 u) w
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 K, J0 ^* Q* ?1 H% |0 i2 z
2.Add one tenth volume of Sodium Acetate buffer to equalize ion concentrations.$ b- P3 `0 d5 W6 T% R9 F
3.Add at least two volumes of cold 100% ethanol; let stand in -20℃ freezer for at least one hour .
3 h* x3 k- H- e! Z9 H1 N4.Centrifuge sample for 15 minutes at highest speed in a 4℃ microcentrifuge .6 Y) [1 w7 x9 m, W- A
5.Remove as much supernatant as possible with a 1 mL micropipet; recentrifuge, then remove the rest with a 200 μl pipet., P6 y7 A7 k  Y+ e' x
6.Add 200 μl of cold 70% ethanol; centrifuge for 5 minutes in a 4 ℃ centrifuge.2 N4 c, P4 l. G  a4 t. H
7.Remove supernatant with a 200 μl pipet; keep for 5 minites at RT. / n2 ^8 K& B2 X8 D7 |) @* q; w& Y
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。