Difference: ProtocolsConjugation (8 vs. 9)

Revision 92018-05-29 - SeanLeonard

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META TOPICPARENT name="ProtocolList"

General Conjugation Protocol

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 Conjugation is a reliable, robust method to transfer plasmids between bacteria. This is a general purpose protocol for conjugation using a DAP (diaminopimelic acid, 0.3 mM)- donor strain with a standard strain. Many steps can be optimized, but this is a good starting point for trying a new strain. For additional resources, concerns, and options, see the end of this document.

Required:

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  • Donor strain that contains your plasmid of interest (this should be a strain like Mu Free Donor or B2155, which contain chromosomal integrated rp4 based transfer machinery). Your plasmid should contain an oriT sequence (known as the origin of transfer, this allows it to be transferred)
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  • Donor strain that contains your plasmid of interest (this should be a strain like Mu Free Donor (MFDpir) or B2155, which contain chromosomal integrated rp4 based transfer machinery). MFDpir can be requested from its creators. Your plasmid should contain an oriT sequence (known as the origin of transfer, this allows it to be transferred)
 
  • Recipient strain
  • Growth media for each individual strain.
  • Non-selective plates containing DAP (0.3 mM) for conjugation. These plates should be made from media that both strains can grow on, and contain no antibiotic. You can set up ~4 conjugation mixtures per plate.
 
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