乙醇沉淀DNA for new guys

chochochocho

; n  w, V- X9 ~/ w& {+ \
) S. ~& l) _5 j8 G' v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。
  Y% s# Z% x- @# }3 JEthanol Precipitation of DNA- t+ }8 O9 i, ?1 A$ z1 E2 D
This procedure allows the concentration of DNA samples from dilute solution and the removal of unwanted salts from DNA samples.
/ O6 ^5 p& s1 d) J' H  I2 A, `Materials
. R* T/ ?; U; I4 K: H. l! j# {) t! u•3M Sodium Acetate buffer, pH 5.29 O( v$ A/ ?: M1 T# j" _, d, P
•Cold 100% Ethanol
* U' f% R9 x9 B5 I0 d& s6 G•Cold 70% Ethanol in sterile dH2O
- P& m8 l8 _5 W# Z0 Q' D•DNA sample6 G7 L# P) S* d% i8 Y7 r! ^" Q
•4 ℃ Microcentrifuge
+ h0 c1 B' E, R( K6 N* O+ `' S
/ }  [1 |1 D$ ^' q7 G* k/ YProcedure
/ a$ `& x2 @: {; h# S2 G1.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).4 ?9 ]6 f# m, m8 A9 B
2.Add one tenth volume of Sodium Acetate buffer to equalize ion concentrations.6 E7 V) B: W  \; g! Z  D; `/ V
3.Add at least two volumes of cold 100% ethanol; let stand in -20℃ freezer for at least one hour .; T# r$ B5 J% y& u5 u
4.Centrifuge sample for 15 minutes at highest speed in a 4℃ microcentrifuge .+ v% a; |' s, q% U3 a) H
5.Remove as much supernatant as possible with a 1 mL micropipet; recentrifuge, then remove the rest with a 200 μl pipet.
: d+ n2 K9 d' l6.Add 200 μl of cold 70% ethanol; centrifuge for 5 minutes in a 4 ℃ centrifuge.
# Y: U3 H; Z3 M4 K5 F7 h7.Remove supernatant with a 200 μl pipet; keep for 5 minites at RT.   W4 g9 H" L! x' i5 Z8 ]4 s, ~; J1 ]
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。