Bacterial transformation

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''Back to [[Protocols]]''
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{{back to protocols}}
==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'''.
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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 [[Molecular Cloning]] for a fuller discussion of both approaches.
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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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[[Chemical transformation buffer comparison]]
[[Chemical transformation buffer comparison]]
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Someone should check out the claims of Nishimura90.  [[User:Tk|tk]] 08:58, 25 September 2007 (EDT)
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Rubidium chloride transformation protocol [http://wheat.pw.usda.gov/~lazo/methods/goldberg/competen.html here]
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Someone should check the claims of 1e10 chemical competence using 10% ethanol and calcium chloride protocols [http://www.ejbiotechnology.info/content/vol10/issue1/full/10/index.html here].
===Electroporation===
===Electroporation===
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#MolecularCloning isbn=0-87969-577-3
#MolecularCloning isbn=0-87969-577-3
# Hanahan91 pmid=1943786
# Hanahan91 pmid=1943786
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# Nishimura90 pmid=2235524
# Hanahan89 US Patent 4,851,348 [[Media:pat4851348.pdf]]
# Hanahan89 US Patent 4,851,348 [[Media:pat4851348.pdf]]
# Jessee90 US Patent 4,981,797  [[Media:pat4981797.pdf]]
# Jessee90 US Patent 4,981,797  [[Media:pat4981797.pdf]]
# Donahue01 US Patent 6,247,369 [[Media:pat6274369.pdf]]
# Donahue01 US Patent 6,247,369 [[Media:pat6274369.pdf]]
# Greenr04 US Patent 6,706,525 [[Media:pat6706525.pdf]]
# Greenr04 US Patent 6,706,525 [[Media:pat6706525.pdf]]
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# Donahue04 US Patent 6,709,854 [[Media:pat6709854.pdf]]
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# Donahue04 US Patent 6,709,854 [[Media:Pat6709854.pdf]]
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# Bloom04 US Patent 6,709,852 [[Media:pat6709852.pdf]]
# Bloom05 US Patent 6,855,494 [[Media:pat6855494.pdf]]
# Bloom05 US Patent 6,855,494 [[Media:pat6855494.pdf]]
# Jessee05 US Patent 6,960,464 [[Media:pat6960464.pdf]]
# Jessee05 US Patent 6,960,464 [[Media:pat6960464.pdf]]
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# Cohen-PNAS-1972 pmid=4559594
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// This appears to be one of the earliest papers on transformation of Escherichia coli.  If you find an earlier paper, please include it here.
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# 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].
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</biblio>
</biblio>
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[[Category:Protocol]]
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[[Category:In vivo]]
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[[Category:Escherichia coli]]

Current revision

back to protocols

Contents

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 -

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

  1. isbn:0-87969-577-3. [MolecularCloning]
  2. Hanahan D, Jessee J, and Bloom FR. . pmid:1943786. PubMed HubMed [Hanahan91]
  3. Nishimura A, Morita M, Nishimura Y, and Sugino Y. . pmid:2235524. PubMed HubMed [Nishimura90]
  4. US Patent 4,851,348 Media:pat4851348.pdf [Hanahan89]
  5. US Patent 4,981,797 Media:pat4981797.pdf [Jessee90]
  6. US Patent 6,247,369 Media:pat6274369.pdf [Donahue01]
  7. US Patent 6,706,525 Media:pat6706525.pdf [Greenr04]
  8. US Patent 6,709,854 Media:Pat6709854.pdf [Donahue04]
  9. US Patent 6,709,852 Media:pat6709852.pdf [Bloom04]
  10. US Patent 6,855,494 Media:pat6855494.pdf [Bloom05]
  11. US Patent 6,960,464 Media:pat6960464.pdf [Jessee05]
  12. Cohen SN, Chang AC, and Hsu L. . pmid:4559594. PubMed HubMed [Cohen-PNAS-1972]
    This appears to be one of the earliest papers on transformation of Escherichia coli. If you find an earlier paper, please include it here.

  13. 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. [Sharma07]
All Medline abstracts: PubMed HubMed
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