CN106916760A - The preparation method and method for transformation of a kind of competent cell - Google Patents
The preparation method and method for transformation of a kind of competent cell Download PDFInfo
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- CN106916760A CN106916760A CN201510984965.3A CN201510984965A CN106916760A CN 106916760 A CN106916760 A CN 106916760A CN 201510984965 A CN201510984965 A CN 201510984965A CN 106916760 A CN106916760 A CN 106916760A
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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Abstract
A kind of preparation method of competent cell, step is as follows：（1）Picking Escherichia coli single bacterium colony, 37 DEG C of 200rpm shaken cultivations are overnight in the test tube equipped with 5ml LB fluid nutrient mediums；The 5ml thalline of culture are all poured into the 1000ml triangular flasks equipped with 100ml LB fluid nutrient mediums, 37 DEG C of shaken cultivation 2h, be 0.5 to OD600 values；（2）During cultured bacterium solution gone into the 50ml centrifuge tubes of ice precooling, 30min is placed on ice；（3）4 DEG C, 4000rpm centrifugation 10min abandon supernatant, and being inverted 1min flows to end solution, reclaims thalline；（4）The precooling 0.1M CaCl of 1/10 times of volume of original bacteria liquid are added in each 50ml centrifuge tube2, the resuspended thalline of pipette tips piping and druming；Ice bath 10min, 4 DEG C, 4000rpm centrifugation 10min reclaim thalline；（5）Repeat step 4 is once；（6）Per 50ml nutrient solutions with the 0.1M CaCl of 2ml ice precoolings2Resuspended, addition final glycerol concentration is 15%-20%；（7）In packing 1.5ml EP pipes, every μ l of pipe 50, -80 DEG C save backup after liquid nitrogen cryopreservation.Competent cell preparation method of the invention is simple, low cost, and high conversion rate.
Description
Technical field
The invention belongs to technical field of molecular biology, and in particular to the preparation method and its method for transformation of a kind of competent cell.
Background technology
Conversion is that exogenous DNA molecule is introduced into acceptor bacterium, is allowed to obtain new inhereditary feature.This is the most basic operating technology of modern molecular biology.Transformation efficiency directly affects the progress and operating efficiency of before and after test, and the state of competent cell is one of most direct, crucial factor of influence transformation efficiency.Generally there are two methods to obtain competent cell, one kind is to be purchased from commercial company, and this competence generally has transformation efficiency higher, 10 are produced per g plasmid DNA8The transformation efficiency of individual transformed clone, but price is costly.Another method is prepared by laboratory oneself.Conventional competent cell preparation method has CaCl at present2With RbCl/KCl methods.The competent cell transformation efficiency of RbCl/KCl methods is higher, but needs special reagent RbCl, CaCl2Method is simple and easy to do, but transformation efficiency is relatively low.In addition to above two method, the Inoue methods and electroporation of super competent cell are also prepared, the condition of culture of Inoue method bacteriums is 18 DEG C, and general laboratory is extremely difficult to.Electroporation requirement electroporation apparatus, common laboratory does not have the equipment.Thus, a kind of transformation efficiency of research is high, and reproducible, the competence preparation method pair suitable for common lab simple to operate is particularly necessary with the research of molecular biology, significant to Molecular Cloning: A Laboratory.
The content of the invention
Regarding to the issue above, the present invention is intended to provide a kind of preparation method of transformation efficiency competent cell high.
A kind of preparation method of competent cell, comprises the following steps：
（1）Picking Escherichia coli single bacterium colony, 37 DEG C of 200rpm shaken cultivations are overnight in the test tube equipped with 5ml LB fluid nutrient mediums；The 5ml thalline of culture are all poured into the 1000ml triangular flasks equipped with 100ml LB fluid nutrient mediums, 37 DEG C of shaken cultivation 2h, be 0.5 to OD600 values；
（2）During cultured bacterium solution gone into the 50ml centrifuge tubes of ice precooling, 30min is placed on ice；
（3）4 DEG C, 4000rpm centrifugation 10min abandon supernatant, and being inverted 1min flows to end solution, reclaims thalline；
（4）The precooling 0.1M CaCl of 1/10 times of volume of original bacteria liquid are added in each 50ml centrifuge tube2, the resuspended thalline of pipette tips piping and druming；Ice bath 10min, 4 DEG C, 4000rpm centrifugation 10min reclaim thalline；
（5）Repeat step 4 is once；
（6）Per 50ml nutrient solutions with the 0.1M CaCl of 2ml ice precoolings2Resuspended, addition final glycerol concentration is 15%-20%；
（7）In packing 1.5ml EP pipes, every μ l of pipe 50, -80 DEG C save backup after liquid nitrogen cryopreservation.
The method for transformation of above-mentioned competent cell, comprises the following steps：
（1）50 μ l competent cells, plus 5 μ l connection products are taken, is gently mixed, 30min is placed on ice；
（2）42 DEG C of heat shock 90s, place 2min on ice；
（3）Plus 800 μ l LB culture mediums, 37 DEG C of 200rpm shaken cultivations 1h；
（4）The rpm of room temperature 4000 is centrifuged 5min, part supernatant is suctioned out, with remaining culture medium suspension thalline；
（5）Bacterium is coated on the solid LB media flat board containing ammonia benzyl；
（6）Flat board is absorbed into surplus liquid in 37 DEG C of positive 1h that place, overnight incubation is inverted.
Competent cell preparation method of the invention is simple, low cost, and high conversion rate.
Specific embodiment
The present invention is described in further details with reference to specific embodiment.
A kind of preparation method of competent cell, comprises the following steps：
（1）Picking Escherichia coli single bacterium colony, 37 DEG C of 200rpm shaken cultivations are overnight in the test tube equipped with 5ml LB fluid nutrient mediums；The 5ml thalline of culture are all poured into the 1000ml triangular flasks equipped with 100ml LB fluid nutrient mediums, 37 DEG C of shaken cultivation 2h, be 0.5 to OD600 values；
（2）During cultured bacterium solution gone into the 50ml centrifuge tubes of ice precooling, 30min is placed on ice；
（3）4 DEG C, 4000rpm centrifugation 10min abandon supernatant, and being inverted 1min flows to end solution, reclaims thalline；
（4）The precooling 0.1M CaCl of 1/10 times of volume of original bacteria liquid are added in each 50ml centrifuge tube2, the resuspended thalline of pipette tips piping and druming；Ice bath 10min, 4 DEG C, 4000rpm centrifugation 10min reclaim thalline；
（5）Repeat step 4 is once；
（6）Per 50ml nutrient solutions with the 0.1M CaCl of 2ml ice precoolings2Resuspended, addition final glycerol concentration is 15%-20%；
（7）In packing 1.5ml EP pipes, every μ l of pipe 50, -80 DEG C save backup after liquid nitrogen cryopreservation.
The method for transformation of above-mentioned competent cell, comprises the following steps：
（1）50 μ l competent cells, plus 5 μ l connection products are taken, is gently mixed, 30min is placed on ice；
（2）42 DEG C of heat shock 90s, place 2min on ice；
（3）Plus 800 μ l LB culture mediums, 37 DEG C of 200rpm shaken cultivations 1h；
（4）The rpm of room temperature 4000 is centrifuged 5min, part supernatant is suctioned out, with remaining culture medium suspension thalline；
（5）Bacterium is coated on the solid LB media flat board containing ammonia benzyl；
（6）Flat board is absorbed into surplus liquid in 37 DEG C of positive 1h that place, overnight incubation is inverted.
Claims (2)
1. a kind of preparation method of competent cell, it is characterised in that comprise the following steps：
（1）Picking Escherichia coli single bacterium colony, 37 DEG C of 200rpm shaken cultivations are overnight in the test tube equipped with 5ml LB fluid nutrient mediums；The 5ml thalline of culture are all poured into the 1000ml triangular flasks equipped with 100ml LB fluid nutrient mediums, 37 DEG C of shaken cultivation 2h, be 0.5 to OD600 values；
（2）During cultured bacterium solution gone into the 50ml centrifuge tubes of ice precooling, 30min is placed on ice；
（3）4 DEG C, 4000rpm centrifugation 10min abandon supernatant, and being inverted 1min flows to end solution, reclaims thalline；
（4）The precooling 0.1M CaCl of 1/10 times of volume of original bacteria liquid are added in each 50ml centrifuge tube2, the resuspended thalline of pipette tips piping and druming；Ice bath 10min, 4 DEG C, 4000rpm centrifugation 10min reclaim thalline；
（5）Repeat step 4 is once；
（6）Per 50ml nutrient solutions with the 0.1M CaCl of 2ml ice precoolings2Resuspended, addition final glycerol concentration is 15%-20%；
（7）In packing 1.5ml EP pipes, every μ l of pipe 50, -80 DEG C save backup after liquid nitrogen cryopreservation.
2. the method for transformation of competent cell prepared by method according to claim 1, it is characterised in that comprise the following steps：
（1）50 μ l competent cells, plus 5 μ l connection products are taken, is gently mixed, 30min is placed on ice；
（2）42 DEG C of heat shock 90s, place 2min on ice；
（3）Plus 800 μ l LB culture mediums, 37 DEG C of 200rpm shaken cultivations 1h；
（4）The rpm of room temperature 4000 is centrifuged 5min, part supernatant is suctioned out, with remaining culture medium suspension thalline；
（5）Bacterium is coated on the solid LB media flat board containing ammonia benzyl；
（6）Flat board is absorbed into surplus liquid in 37 DEG C of positive 1h that place, overnight incubation is inverted.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109852566A (en) * | 2019-03-20 | 2019-06-07 | 徐州医科大学 | A kind of preparation method and method for transformation of Electroporation-competent cells |
| CN110484552A (en) * | 2019-08-06 | 2019-11-22 | 上海药明生物技术有限公司 | The preparation method of non-animal derived property Plasmid DNA |
| CN110777106A (en) * | 2019-11-29 | 2020-02-11 | 宁波酶赛生物工程有限公司 | Preparation method of electrocompetent cell |
| CN111534464A (en) * | 2020-05-07 | 2020-08-14 | 内蒙古工业大学 | Preparation method of Xanthomonas campestris competent cells |
-
2015
- 2015-12-25 CN CN201510984965.3A patent/CN106916760A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109852566A (en) * | 2019-03-20 | 2019-06-07 | 徐州医科大学 | A kind of preparation method and method for transformation of Electroporation-competent cells |
| CN110484552A (en) * | 2019-08-06 | 2019-11-22 | 上海药明生物技术有限公司 | The preparation method of non-animal derived property Plasmid DNA |
| CN110777106A (en) * | 2019-11-29 | 2020-02-11 | 宁波酶赛生物工程有限公司 | Preparation method of electrocompetent cell |
| CN110777106B (en) * | 2019-11-29 | 2023-02-03 | 宁波酶赛生物工程有限公司 | Preparation method of electrocompetent cell |
| CN111534464A (en) * | 2020-05-07 | 2020-08-14 | 内蒙古工业大学 | Preparation method of Xanthomonas campestris competent cells |
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