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电转影响因素及原理 已有3人参与
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影响电转的因素及如何影响的，已经电转了很多次还是没有克隆长出来，想了解下一些影响因素及原理，然后好去继续实验 发自小木虫Android客户端 |
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- 【原创】非变性凝胶电泳原理及应用实例
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zhouxfd
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【答案】应助回帖
感谢参与，应助指数 +1
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Media SOB 2% tryptone 0.5% yeast extract 10 mM NaCl 2.5 mM KCl 10 mM MgCl2 10 mM MgSO4 SOC SOB + 20 mM glucose Appropriate Antibiotics for Your Application Antibiotics for Plasmid selection Antibiotic Working Concentration Ampicillin 100 μg/ml Carbenicillin 100 μg/ml Chloramphenicol 33 μg/ml Kanamycin 30 μg/ml Streptomycin 25 μg/ml Tetracycline 15 μg/ml Sterile 10% glycerol (can be autoclaved) is needed for the washes. The volume of 10% glycerol needed is 2X the culture volume (for example, a 500 ml culture requires 1L of 10% glycerol). Procedure (for 2, 250 ml cultures) Inoculate 1 colony from a fresh plate of the strain to be made electrocompetent into 10 ml of SOB in a 125 ml flask and incubate for 16-18 hours at 37oC and 250 rpm. Have ready 2, 1 L flasks containing 250 ml each of SOB pre-warmed to 37°C. Add two drops of the overnight culture to each of the flasks. Shake at 37°C and 250 rpm until the cultures reach an OD600 of 0.5-0.7. Be sure to turn on centrifuge and cool rotor to 4°C well in advance of harvesting cells. Be sure to place 1 L of 10% glycerol on ice well in advance of harvesting cells Place cultures on ice for 15 minutes. From this point on the cultures must be kept ice cold. Pour each 250 ml culture into chilled 500 ml (or 1000 ml) centrifuge bottles. Centrifuge at 5000 rpm for 10 min. Pour off the supernatant and aspirate any residual broth. Add 250 ml of glycerol to each of the centrifuge bottles and completely suspend the cells by pipetting up and down. Centrifuge at 5000 rpm for 10 min. Pour off the supernatant, it is not necessary to aspirate. Completely suspend the cells in 250 ml glycerol and re-centrifuge. Pour off the supernatant and suspend the cells in the residual glycerol by pipetting up and down. At this point you can electroporate or freeze the cells away. To freeze, add 100 microliters of the culture to microcentrifuge tubes on ice. Once you have used all of the culture, transfer the tubes to dry ice for 10 minutes. Once the cultures are frozen, transfer them to a -80°C freezer. The cultures should be good for >6 months. Electroporation Protocol The electroporation protocol will vary depending on the strain so this protocol may need to be optimized. For control electroporation dilute pUC19 to 10 pg/μl with Milli-Q water. Turn on electroporator and set to 1.7-2.5 kv (optimize for strain), 200 ohms and 25 μF. Place recovery SOC in 37°C water bath. Pre-warm LB-antibiotic plates at 37°C. Thaw cells on ice for 10 min or use freshly made cells. Place appropriate number of microcentrifuge tubes and 1 mm-electroporation cuvettes on ice. Flick the tube containing cells a few times to mix and add 25 μl to the microcentrifuge tubes. Add 1 μl of a 10 pg/μl DNA solution (in DI water) to the cells in the microcentrifuge tube. Transfer the DNA-cell mixture to the cold cuvette, tap on countertop 2X, wipe water from exterior of cuvette and place in the electroporation module and press pulse (don’t hold the button down). Immediately add 975 μl of 37°C SOC, mix by pipetting up and down once and transfer to a 15 ml-falcon tube. Rotate in the 37°C incubator for 1 h. Make appropriate dilutions. When using 10 pg of DNA, make two dilutions: Dilute 10 μl cells into 990 μl SOC and plate 100 μl. (1000-fold dilution) Dilute 100 μl cells into 900 μl SOC and plate 100 μl. (100-fold dilution) Incubate overnight at 37°C. Calculation: If the culture was diluted 1000-fold when plated, the total cfu per ml is 1000 times the number of colonies counted. The cfu is divided by the amount of pUC19 (10 pg per ml) cfu/ μg = (colonies counted*1000) / (0.00001 μg pUC19) |
5楼2017-08-02 01:03:34
【答案】应助回帖
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感谢参与，应助指数 +1
西门吹雪170: 金币+6, MolEPI+1, 鼓励回帖交流 2017-08-02 07:45:31
感谢参与，应助指数 +1
西门吹雪170: 金币+6, MolEPI+1, 鼓励回帖交流 2017-08-02 07:45:31
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电转原理： 电转化法是利用瞬间高压在细胞上打孔，因而需用冰冷的超纯水多次洗涤处于对数生长前期的细胞，以使细胞悬浮液中应含有尽量少的导电离子。转化效率为109～1010 转化子/礸 DNA；对于热激法，是利用冰冷的CaCl2处理对数生长期的细胞，可以诱导其产生短暂的“感受态‘，易于摄取外源DNA。转化效率为106 ～107转化子/礸 DNA。 影响因素： ⑴、细胞的生长状态和密度 最好从-70℃或-20℃甘油保存的菌种中直接转接用于制备感受态细胞的菌液。不要用已 经过多次转接，及贮存在4℃的培养菌液。细胞生长密度以每毫升培养液中的细胞数在5×107个左右为佳。即应用对数期或对数生长前期的细菌，可通过测定培养液的OD600控制。对TG1菌株，OD600为0.5时，细胞密度在5×107个/ml左右。(应注意OD600值与细胞数之间的关系随菌株的不同而不同)。密度过高或不足均会使转化率下降。 此外，受体细胞一般应是限制-修饰系统缺陷的突变株，即不含限制性内切酶和甲基化酶的突变株。并且受体细胞还应与所转化的载体性质相匹配。 ⑵、质粒dna的质量和浓度 用于转化的质粒DNA应主要是超螺旋态的，转化率与外源DNA的浓度在一定范围内成正比，但当加入的外源DNA的量过多或体积过大时，则会使转化率下降。一般地，DNA溶液的体积不应超过感受态细胞体积的5%，1ng的cccDNA即可使50ul的感受态细胞达到饱和。 对于以质粒为载体的重组分子而言，分子量大的转化效率低，实验证明，大于30kb的重组质粒将很难进行转化。此外，重组DNA分子的构型与转化效率也密切相关，环状重组质粒的转化率较分子量相同的线性重组质粒高10~100倍，因此重组DNA大都构成环状双螺旋分子。 ⑶、试剂的质量 所用的CaCl2等试剂均需是最高纯度的，并用最纯净的水配制，最好分装保存于4℃。 ⑷、防止杂菌和杂DNA的污染 整个操作过程均应在无菌条件下进行，所用器皿，如离心管，移液枪头等最好是新的，并经高压灭菌处理。所有的试剂都要灭菌，且注意防止被其它试剂、DNA酶或杂DNA所污染，否则均会影响转化效率或杂DNA的转入。 ⑸、整个操作均需在冰上进行，不能离开冰浴，否则细胞转化率将会降低。 |
2楼2017-08-01 22:38:46
3楼2017-08-01 23:23:46
上一条不小心发出去了 一楼CaCl2都出来了 这是电转 哪来的CaCl2啊 讲道理电转的效率一般情况下很高 楼主最好把感受态制备和电转流程写一下 这样便于别人找问题在哪里哦 发自小木虫IOS客户端 |
4楼2017-08-01 23:26:32
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