Bacterial transformation: Difference between revisions
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==Introduction== | ==Introduction== | ||
Transformation is the process of introducing foreign DNA (e.g plasmids, BAC) into a bacterium. Bacterial cells into which foreign DNA can be transformed are called '''competent'''. Some bacteria are naturally competent (e.g ''B. subtilis''), whereas others such as ''E. coli'' are not naturally competent. Non-competent cells can be made competent and then transformed via one of two main approaches; '''chemical transformation''' and '''electroporation'''. | Transformation is the process of introducing foreign DNA (e.g plasmids, BAC) into a bacterium. Bacterial cells into which foreign DNA can be transformed are called '''competent'''. Some bacteria are naturally competent (e.g ''B. subtilis''), whereas others such as ''E. coli'' are not naturally competent. Non-competent cells can be made competent and then transformed via one of two main approaches; '''chemical transformation''' and '''electroporation'''. | ||
There are advantages and disadvantages to both transformation methods. In general, chemical transformation is less prone to error and faster however electroporation produces a higher transformation efficiency (fraction of transformed cells that actually uptake the foreign DNA). See [[ | There are advantages and disadvantages to both transformation methods. In general, chemical transformation is less prone to error and faster however electroporation produces a higher transformation efficiency (fraction of transformed cells that actually uptake the foreign DNA). See [[Cloning]] for a fuller discussion of both approaches. | ||
==Protocols== | ==Protocols== | ||
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# Cohen-PNAS-1972 pmid=4559594 | # Cohen-PNAS-1972 pmid=4559594 | ||
// This appears to be one of the earliest papers on transformation of Escherichia coli. If you find an earlier paper, please include it here. | // This appears to be one of the earliest papers on transformation of Escherichia coli. If you find an earlier paper, please include it here. | ||
# Sharma07 Sharma AD, Singh J, Gill PK; Ethanol mediated enhancement of bacterial transformation, Electronic Journal of Biotechnology (2007), 10(1), DOI: 10.2225/vol10-issue1-fulltext-10, [http://www.ejbiotechnology.info/content/vol10/issue1/full/10/index.html here]. | |||
</biblio> | </biblio> | ||
Latest revision as of 20:45, 29 December 2011
| back to protocols | ||
Introduction
Transformation is the process of introducing foreign DNA (e.g plasmids, BAC) into a bacterium. Bacterial cells into which foreign DNA can be transformed are called competent. Some bacteria are naturally competent (e.g B. subtilis), whereas others such as E. coli are not naturally competent. Non-competent cells can be made competent and then transformed via one of two main approaches; chemical transformation and electroporation.
There are advantages and disadvantages to both transformation methods. In general, chemical transformation is less prone to error and faster however electroporation produces a higher transformation efficiency (fraction of transformed cells that actually uptake the foreign DNA). See Cloning for a fuller discussion of both approaches.
Protocols
OpenWetWare already has a number of protocols relating to bacterial transformation but more are always welcome.
If you use a variant on one of these protocols please feel free to add a link to your protocol from one of these pages so other users can find a protocol that works for them. Additionally, if anyone uses the Innoe or Hanahan high-efficiency protocols, then please add protocols here.
Chemical transformation
If you plan on doing a chemical transformation, then you should see these pages -
- Preparing chemically competent cells
- Preparing TSS buffer
- Transforming chemically competent cells
- Preparing chemically competent cells (Inoue)
- Transforming chemically competent cells (Inoue)
- TOP10 chemically competent cells
Chemical transformation buffer comparison
Someone should check out the claims of Nishimura90. tk 08:58, 25 September 2007 (EDT)
Rubidium chloride transformation protocol here
Someone should check the claims of 1e10 chemical competence using 10% ethanol and calcium chloride protocols here.
Electroporation
If you plan on using electroporation, then see these pages -
References
- ISBN:0-87969-577-3
- Hanahan D, Jessee J, and Bloom FR. Plasmid transformation of Escherichia coli and other bacteria. Methods Enzymol. 1991;204:63-113. DOI:10.1016/0076-6879(91)04006-a |
- Nishimura A, Morita M, Nishimura Y, and Sugino Y. A rapid and highly efficient method for preparation of competent Escherichia coli cells. Nucleic Acids Res. 1990 Oct 25;18(20):6169. DOI:10.1093/nar/18.20.6169 |
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US Patent 4,851,348 Media:pat4851348.pdf
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US Patent 4,981,797 Media:pat4981797.pdf
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US Patent 6,247,369 Media:pat6274369.pdf
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US Patent 6,706,525 Media:pat6706525.pdf
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US Patent 6,709,854 Media:Pat6709854.pdf
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US Patent 6,709,852 Media:pat6709852.pdf
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US Patent 6,855,494 Media:pat6855494.pdf
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US Patent 6,960,464 Media:pat6960464.pdf
- Cohen SN, Chang AC, and Hsu L. Nonchromosomal antibiotic resistance in bacteria: genetic transformation of Escherichia coli by R-factor DNA. Proc Natl Acad Sci U S A. 1972 Aug;69(8):2110-4. DOI:10.1073/pnas.69.8.2110 |
This appears to be one of the earliest papers on transformation of Escherichia coli. If you find an earlier paper, please include it here.
-
Sharma AD, Singh J, Gill PK; Ethanol mediated enhancement of bacterial transformation, Electronic Journal of Biotechnology (2007), 10(1), DOI: 10.2225/vol10-issue1-fulltext-10, here.
