A comparison and optimization of methods and factors affecting the transformation of Escherichia coli
DNA manipulation routinely requires competent bacteria that can be made using one of numerous methods. To determine the best methods, we compared four commonly used chemical methods (DMSO, MgCl2–CaCl2, CaCl2 and Hanahan's methods) on frequently used Escherichia coli (E. coli) strains: DH5α, XL-...
| Main Authors: | , , , |
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| Format: | Journal Article |
| Language: | English |
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2015
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| Online Access: | https://hdl.handle.net/10356/81298 http://hdl.handle.net/10220/39215 |
| _version_ | 1811683488345096192 |
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| author | Verma, Chandra Shekhar Lane, David P. Chan, Weng-Tat Gan, Samuel Ken-En |
| author2 | School of Biological Sciences |
| author_facet | School of Biological Sciences Verma, Chandra Shekhar Lane, David P. Chan, Weng-Tat Gan, Samuel Ken-En |
| author_sort | Verma, Chandra Shekhar |
| collection | NTU |
| description | DNA manipulation routinely requires competent bacteria that can be made using one of numerous methods. To determine the best methods, we compared four commonly used chemical methods (DMSO, MgCl2–CaCl2, CaCl2 and Hanahan's methods) on frequently used Escherichia coli (E. coli) strains: DH5α, XL-1 Blue, SCS110, JM109, TOP10 and BL21-(DE3)-PLysS. Hanahan's method was found to be most effective for DH5α, XL-1 Blue and JM109 strains (P<0.05), whilst the CaCl2 method was best for SCS110, TOP10 and BL21 strains (P<0.05). The use of SOB (super optimal broth) over LB [Luria–Bertani (broth)] growth media was found to enhance the competency of XL-1 Blue (P<0.05), dampened JM109′s competency (P<0.05), and had no effect on the other strains (P>0.05). We found no significant differences between using 45 or 90 s heat shock across all the six strains (P>0.05). Through further optimization by means of concentrating the aliquots, we were able to get further increases in transformation efficiencies. Based on the optimized parameters and methods, these common laboratory E. coli strains attained high levels of TrE (transformation efficiency), thus facilitating the production of highly efficient and cost-effective competent bacteria. |
| first_indexed | 2024-10-01T04:13:32Z |
| format | Journal Article |
| id | ntu-10356/81298 |
| institution | Nanyang Technological University |
| language | English |
| last_indexed | 2024-10-01T04:13:32Z |
| publishDate | 2015 |
| record_format | dspace |
| spelling | ntu-10356/812982023-02-28T16:58:56Z A comparison and optimization of methods and factors affecting the transformation of Escherichia coli Verma, Chandra Shekhar Lane, David P. Chan, Weng-Tat Gan, Samuel Ken-En School of Biological Sciences Competent bacteria DMSO method Hanahan’s method CaCl2 method Heat shock DNA manipulation routinely requires competent bacteria that can be made using one of numerous methods. To determine the best methods, we compared four commonly used chemical methods (DMSO, MgCl2–CaCl2, CaCl2 and Hanahan's methods) on frequently used Escherichia coli (E. coli) strains: DH5α, XL-1 Blue, SCS110, JM109, TOP10 and BL21-(DE3)-PLysS. Hanahan's method was found to be most effective for DH5α, XL-1 Blue and JM109 strains (P<0.05), whilst the CaCl2 method was best for SCS110, TOP10 and BL21 strains (P<0.05). The use of SOB (super optimal broth) over LB [Luria–Bertani (broth)] growth media was found to enhance the competency of XL-1 Blue (P<0.05), dampened JM109′s competency (P<0.05), and had no effect on the other strains (P>0.05). We found no significant differences between using 45 or 90 s heat shock across all the six strains (P>0.05). Through further optimization by means of concentrating the aliquots, we were able to get further increases in transformation efficiencies. Based on the optimized parameters and methods, these common laboratory E. coli strains attained high levels of TrE (transformation efficiency), thus facilitating the production of highly efficient and cost-effective competent bacteria. ASTAR (Agency for Sci., Tech. and Research, S’pore) Published version 2015-12-23T08:17:08Z 2019-12-06T14:27:47Z 2015-12-23T08:17:08Z 2019-12-06T14:27:47Z 2013 Journal Article Chan, W., Verma, C., Lane, D., & Gan, S. (2013). A comparison and optimization of methods and factors affecting the transformation of Escherichia coli. Bioscience Reports, 33(6), 931-937. 0144-8463 https://hdl.handle.net/10356/81298 http://hdl.handle.net/10220/39215 10.1042/BSR20130098 24229075 en Bioscience Reports © 2013 The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Licence (CC-BY) (http://creativecommons.org/licenses/by/3.0/) which permits unrestricted use, distribution and reproduction in any medium, provided the original work is properly cited. 13 p. application/pdf |
| spellingShingle | Competent bacteria DMSO method Hanahan’s method CaCl2 method Heat shock Verma, Chandra Shekhar Lane, David P. Chan, Weng-Tat Gan, Samuel Ken-En A comparison and optimization of methods and factors affecting the transformation of Escherichia coli |
| title | A comparison and optimization of methods and factors affecting the transformation of Escherichia coli |
| title_full | A comparison and optimization of methods and factors affecting the transformation of Escherichia coli |
| title_fullStr | A comparison and optimization of methods and factors affecting the transformation of Escherichia coli |
| title_full_unstemmed | A comparison and optimization of methods and factors affecting the transformation of Escherichia coli |
| title_short | A comparison and optimization of methods and factors affecting the transformation of Escherichia coli |
| title_sort | comparison and optimization of methods and factors affecting the transformation of escherichia coli |
| topic | Competent bacteria DMSO method Hanahan’s method CaCl2 method Heat shock |
| url | https://hdl.handle.net/10356/81298 http://hdl.handle.net/10220/39215 |
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