超级感受态
实验步骤：
1、Pick a single bacterial colony (2-3 mm in diameter) from a plate that has been incubated for 16-20 hours at 37oC. Transfer the colony into 25 ml of LB broth or SOB medium in a 250-ml flask. Incubate the culture for 6-8 hours at 37oC with vigorous shaking (250-300 rpm).
2、接种（1-10ml）于250mL SOB，18-22（适度转速）培养⾄OD＝0.6。
3、菌液置冰上10分钟。
4、4度2500g离⼼10分钟。
5、⼩⼼⽤80mL预冷Inouebuffer重悬细胞。
6、菌液置冰上10分钟。
7、4度2500g离⼼10分钟。
8、⼩⼼⽤20mL预冷Inouebuffer重悬细胞。
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9、加⼊DMSO⾄终浓度7％。
10、置冰上10分钟。
11、分装，液氮速冻。
12、冻存。
所需试剂：
0.5mol／L 的Hepes（PH6.7）（⽤KOH调PH值）（过滤灭菌）（负20度分装保存）
Inoue buffer：（100ml）（负20°保存）
1.088g MnCl
2.4H2O
0.166g CaCl2
1.865g KCl2
溶于80ml⽔中，加⼊2ml 0.5ml／L Hepes（PH6.7），最后定容到100ml。（过滤灭菌）SOB Medium
地下消火栓2% (w/v) bacto tryptone
0.5% (w/v) yeast extract
10 mM NaCl
2.5 mM KCl
10 mM MgCl2
10 mM MgSO4
pH 6.7 - 7.0
Note:
Competent cells are fragile (cell wall is thought to be weakened), therefore treat the cells gently when preparing the cells. Do not vortex or pippette up and down to resuspend the cells. Do not spi适合的英文
n the cells at too great a speed (spinning down at 5000g will cau some cells to ly).
Always keep the cells chilled when making competent cell. Do not let them warm up.
Freezing the cells appear to make cells more competent.中国黄梅戏
Some 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.
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).
Heat shock time should be determined for different strains of cell. For DH5alpha or JM109 u 30-45 c. For BL21 u 120 c.
Deactivate liga prior to transformation. Liga may reduce transformation efficiency.
Diluting the ligation mixture (~5x) can also increa transformation efficiecy by reducing the amount of
reagents/contaminants that may affect transformation. Likewi it has been suggested that phenol/chloroform treatment may also increa efficiency, but it is probably too much trouble to bother trying.
The DNA added should not be more then 5% of the volume of competent cells ud. The final DNA concentration should not exceed 5 ng/ul.
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. Transformation efficiency has a roughly inver 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.孙悟空故事
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).
For 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 antibio
tic ud for lection - 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 esntial).
Plating cells - dry 1.5% agar plates (expod upside down) at 37 °C for 2-4 hours just before u, the plate should be able to soak up to 0.8-1 ml of media when plating. For blue-white lection, it is not necessary to make X-gal plate, just add X-gal + IPTG direct to cells, mix and then plate.
Fn键是什么意思Detergents may be detrimental to the transformability of the competent cells, therefore the glassware ud for making competent cells should not be washed with detergents. Polycarbonate flask may also be ud instead of glass flask. DMSO can dissolve polystyrene, therefore u polypropylene tubes.
When cloning difficult and less stable quence (e.g palindrome, repeats, LTR quences), it helps to grow transform cells at lower temperatures (25-30 °C or颜回
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room temperature) in very rich media (e.g. Terrific Broth). Also terminate growth before reaching late stationary growth pha when grown in liquid media (i.e harvest cells at OD550 between 1 and 2). U of stabilizing strain is also uful. There are other methods of making competent cells - e.g. Ca
Cl2 method, RbCl method which is more effective than CaCl2 method. Electroporation is suppod to give higher efficiency (up to 1010 transformants per ug plasmid claimed), but for the simple cloning that we do, its u is not warranted (and it's more expensive, more trouble than it's worth, etc.).
If 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 li" and "Better competent cells"
It 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):
本⽂引⾃：Hanahan et al, Methods in Enzymology 204, 63
