innove法制感受态.doc

实验步骤：1、Inoculate from an overnight grown in LB.从培养过夜的LB平板上挑取单菌落 。2、Grow in 250 ml SOB at 18 until OD600= 0.6.(0.3)接种于250ml SOB，18度培养至OD0.6。3、On ice for 10 minutes.菌液置冰上10分钟。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 4.4度2500g离心10分钟。5、Resuspend cells gently in 80 ml of ice cold TB.小心用80ml预冷TB重悬细胞。6、On ice for 10 minutes.(30min)菌液置冰上10分钟。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 4.4度2500g离心10分钟。8、Resuspend cells gently in 20 ml of ice cold TB.小心用20ml预冷TB重悬细胞。9、Add DMSO to a final concentration of 7%.加入DMSO至终浓度7%。10、Place on ice for 10 minutes.置冰上10分钟。11、Aliquot into 1-2 ml and freeze in liquid nitrogen.分装，液氮速冻 。12、Store in liquid nitrogen.冻存。SOB Medium and TB (Transformation Buffer)SOB2% (w/v) bacto tryptoneTB0.5% (w/v) yeast extract10 mMPipes10 mMNaCl55 mMMnCl22.5 mMKCl15 mMCaCl210 mMMgCl2250 mMKCl10 mMMgSO4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 MnCl2thanks to Markus Schneemann for the tip!pH 6.7 - 7.0Note: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).Always keep the cells chilled when making competent cell.Do not let them warm up.Freezing the cells appear to make cells more competent.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 doesnt appear to work with BL21,so just grow the cells at 30 or 37 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 use 30-45 sec.For BL21 use 120 sec.Deactivate ligase prior to transformation.Ligase may reduce transformation efficiency.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.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/l.The method above should give a transformation efficiency of more than 108 cfu per g of plasmid DNA (pUC or pBluescripts)with over 109 cfu possible.Transformation 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.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 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).Plating cells-dry 1.5% agar plates (exposed upside down)at 37 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.Detergents 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.When cloning difficult and less stable sequence (e.g palindrome,repeats,LTR sequences),it helps to grow transform cells at lower temperatures (25-30 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 OD550between 1 and 2).Use of stabilizing strain is also useful.There are other methods of making competent cells-e.g.CaCl2method,RbCl method which is more effective than CaCl2method.Electroporation is supposed to give higher efficiency (up to 1010 transformants per g plasmid claimed),but for the simple cloning that we do,its use is not warranted (and its more expensive,more trouble than its 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 ltra-competent E.coli and Better competent cellsIt 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):Method for preparation of ultra-competent cells for frozen storage materials:1. 0.5M PIPES (pH 6.7) 100mLDissolve 15.1g PIPES in 80mL pure H2O, adjust the pH to pH 6.7 with 5M KOH2. Inoue transformation buffer 200mL (chilled to 0 before use)MnCl24H2O 2.176gCaCl22H2O 0.440gKCl 3.730gPIPES (0.5M, pH 6.7) 4.000mLDissolve MnCl24H2O, CaCl22H2O, KCl in 160mL pure H2O and then add 4mLAll reagents should be sterile and store frozen at -203.DMSO4.SOB mediumSOB培养基配制每升培养基，在950ml去离子水中加入：胰化蛋白胨 20g酵母提取物 5gNaCl 0.5 g摇动容器使溶质完全溶解。加10ml 250mmolL KCl溶液(将1.86g KCl用100ml去离子水溶解即配成250mmol/l KCl溶液)。用5mol/L NaOH 调pH值至7.0。用去离子水定容至1L。在15 psi(1.05kgcm2)高压下蒸汽灭菌20min。该溶液在使用前，加入5ml灭菌的2 mol/l MgCl22molL MgCl2溶液的配制方法如下：用90ml去离子水溶解19g MgCl2，用去离子水调整体积为100ml，在15psi(1.05kgcm2)高压下蒸汽灭菌20min。（可直接分为三瓶配好灭菌，每瓶200ml）Procedure:1.Streak bacteria stock to the appropriate growth plate and incubated at 37 for 16-20h2.Pick a single colony to 25mL LB media in 250mL flask and incubated at 37, 250rpm, 6-8hSeed 10mL/4mL/2mL in three flask of saturated grown bacteria in 200 SOB media, 18-22, 250rpm3.When the OD600=0.55(0.40.55 is OK) (45min/each detect), transfer the culture to an ice-water bath for 10min4.Harvest the cells 2500g, 10min, 4, pour