Inoue法制备大肠杆菌超级感受态细胞
/ O* v" j8 v! g实验步骤：
( f( t9 E8 \! F" ~1、 Inoculate from an overnight grown in LB.从培养过夜的LB平板上挑取单菌落 。
6 O" Z( j0 d5 i& q! E8 h' M$ a2、 Grow in 250 ml "SOB" at 18C until OD600 = 0.6. (0.3) 接种于250mL SOB，18度培养至OD＝0.6。
7 a# r0 N/ R. w7 f5 @3、 On ice for 10 minutes. 菌液置冰上10分钟。
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4、 Spin at 2500 x g (5000 rpm in a Sorvall GSA or 3000 rpm in a Beckman J-6B centrifuge) for 10 min. at 4C. 4度2500g离心10分钟。
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5、 Resuspend cells gently in 80 ml of ice cold "TB".小心用80mL预冷TB重悬细胞。
$ q; X, F. m& i: ~" }0 `" F. m6、 On ice for 10 minutes. (30min) 菌液置冰上10分钟。
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7、 Spin at 2500 x g (5000 rpm in a Sorvall GSA, 5500 rpm in a Sorvall SS-34, or 3000 rpm in a Beckman J-6B centrifuge) for 10 min. at 4C. 4度2500g离心10分钟。
% U# G3 `/ T0 g* ]7 |" q$ Q8、 Resuspend cells gently in 20 ml of ice cold "TB".小心用20mL预冷TB重悬细胞。
" U _" u$ y2 c; A9、 Add DMSO to a final concentration of 7%. 加入DMSO至终浓度7％。
2 o5 ~6 Y' l5 H# ]- j* Y# J10、 Place on ice for 10 minutes. 置冰上10分钟。
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11、 Aliquot into 1-2 ml and freeze in liquid nitrogen.分装，液氮速冻 。
+ _& e+ g" _" z. n) @* e. |. }12、 Store in liquid nitrogen. 冻存。
4 l$ j/ v' ]; _ wSOB Medium and TB (Transformation Buffer)+ B! a1 i6 [+ p
SOB/ W7 \9 m2 A: b+ D" M
| 2% (w/v) bacto tryptone; Q7 X7 a; M4 V9 m( K5 {8 @* ^
| TB
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0.5% (w/v) yeast extract$ V% U$ P8 [- o3 @+ A8 b) N
| 10 mM Pipes$ Z# q1 x0 e* o
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10 mM NaCl
1 V3 j3 o3 D M5 t( X | 55 mM MnCl2
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2.5 mM KCl% P* y! L( K% e c- t5 c
| 15 mM CaCl2: r1 p1 n6 e( I% Y5 [0 ~/ \
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10 mM MgCl2
. U" T; \: d& B0 o! a7 w | 250 mM KCl0 e& ?6 q9 g' b0 h
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10 mM MgSO42 K6 u) V) q$ `) j" m9 `
| 在加入MnCl2之前先用5N KOH调pH值到6.7，adjust pH to 6.7 with 5N KOH prior to adding the MnCl2
0 k' n B+ {" d% @$ x: Lthanks to Markus Schneemann for the tip!
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pH 6.7 - 7.0; R7 H3 X0 h4 P
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Competent cells are fragile (cell wall is thought to be weakened), therefore treat the cells gently when preparing these cells. Do not vortex or pippette up and down to resuspend the cells. Do not spin the cells at too great a speed (spinning down at 5000g will cause some cells to lyse).
0 g2 {5 L p8 C. |$ t& d6 cAlways keep the cells chilled when making competent cell. Do not let them warm up.
+ b8 o2 r" x$ w8 CFreezing the cells appear to make cells more competent.
: R: ?& B0 [/ ?6 Z4 mSome cell strains may work better than others (DH5alpha works well in my hand). Note also that some cells (e.g. HB101) has greater recombination activity than others.
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This method doesn't appear to work with BL21, so just grow the cells at 30 or 37 °C when making BL21 competent cells. However, it has been suggested that the efficiency of BL21 prepared using Inoue method may be improved by treating it with DTT before freezing (add to 3.5% v/v of a 2.2M DTT, 10mM KAc pH6 solution and incubate 10 minutes on ice).
5 A0 Y/ ~8 C4 V% ~: l! I6 hHeat shock time should be determined for different strains of cell. For DH5alpha or JM109 use 30-45 sec. For BL21 use 120 sec.
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Deactivate ligase prior to transformation. Ligase may reduce transformation efficiency.
7 T; G2 C$ S: Z9 q- i2 \Diluting the ligation mixture (~5x) can also increase transformation efficiecy by reducing the amount of reagents/contaminants that may affect transformation. Likewise it has been suggested that phenol/chloroform treatment may also increase efficiency, but it is probably too much trouble to bother trying.
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The DNA added should not be more then 5% of the volume of competent cells used. The final DNA concentration should not exceed 5 ng/ul.
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The method above should give a transformation efficiency of more than 108 cfu per ug of plasmid DNA (pUC or pBluescripts) with over 109 cfu possible.
) i$ Z2 U/ D1 ]( s. K- VTransformation efficiency has a roughly inverse relationship with the size of plasmids. Cells with deoR mutaion (e.g. DH5alpha) can improved the transformation of large plasmid. Relaxed plasmids has ~3/4 of the transformation efficiency of supercoiled plasmid.
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2 different plasmids can be transformed at the same time, or one after another. But they must be compatible (they cannot have the same replicon).
; z) s( [2 d( vFor routine transformation whereby efficiency of transformation is of no import, some of the steps may be shorten or omitted. For example, heat-shock step may be unnecessary and recovery incubation time at 37 °C can be reduced or omitted (but do note that this may depends on the antibiotic used for selection - for ampicillin-type antibiotics the incubation time is not really that important, therefore you can plate the cells straight after heat-shock if you wish. for other antibiotics, however, the incubation time may be essential).
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Plating cells - dry 1.5% agar plates (exposed upside down) at 37 °C for 2-4 hours just before use, the plate should be able to soak up to 0.8-1 ml of media when plating. For blue-white selection, it is not necessary to make X-gal plate, just add X-gal + IPTG direct to cells, mix and then plate.
! t3 D! f j* @2 zDetergents may be detrimental to the transformability of the competent cells, therefore the glassware used for making competent cells should not be washed with detergents. Polycarbonate flask may also be used instead of glass flask. DMSO can dissolve polystyrene, therefore use polypropylene tubes.
) K$ e" G, x' J6 HWhen cloning difficult and less stable sequence (e.g palindrome, repeats, LTR sequences), it helps to grow transform cells at lower temperatures (25-30 °C or room temperature) in very rich media (e.g. Terrific Broth). Also terminate growth before reaching late stationary growth phase when grown in liquid media (i.e harvest cells at OD550 between 1 and 2). Use of stabilizing strain is also useful.
6 [' s, q# g4 I9 f* l! xThere are other methods of making competent cells - e.g. CaCl2 method, RbCl method which is more effective than CaCl2 method. Electroporation is supposed to give higher efficiency (up to 1010 transformants per ug plasmid claimed), but for the simple cloning that we do, its use is not warranted (and it's more expensive, more trouble than it's worth, etc.).
+ O+ A \7 N6 u% H4 F9 D5 d% wIf a cooling shaker is not available - grow the cells at room temperature. More discussions on making competent cell as well as references can be found in TIBS articles "Preparing ultra-competent E.coli" and "Better competent cells"
# |& D# W o7 kIt is also possible to transform cells straight from plate. It is convenient but you should expect low efficiency. See the following reference for more details (as well as more information on competent cells and other protocols):
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本文引自：Hanahan et al, Methods in Enzymology 204, 63
