Molecular Cloning Fourth Edition, A Laboratory Manual, by Michael R. Green and Joseph Sambrook

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Transfer of the Reporter Vector into BAC/RecA Cells and Selection of Cointegrates

(Protocol summary only for purposes of this preview site)

Recombination of the reporter vector with the BACleading to the modified BACis facilitated by the presence of RecA, and recombinants are selected for by growth in the presence of chloramphenical, ampicillin, and tetracycline. Only bacteria containing correctly modified BACs and copies of pSV1.RecA will be selected. Unmodified BACs (i.e., those lacking a pLD53.SC2/A-box insert) are eliminated by exposure to ampicillin. Free reporter plasmid remaining in the BAC host bacteria will also be eliminated, because this vector requires the protein to replicate. The cointegrates are selected by growth at high temperature, thereby eliminating the RecA plasmid. Successful modification of the BACformation of the cointegrateis confirmed in separate amplification reactions.


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(Limited time special offer.) Molecular Cloning Fourth Edition, A Laboratory Manual, by Michael R. Green and Joseph Sambrook
Molecular Cloning Fourth Edition, A Laboratory Manual, by Michael R. Green and Joseph Sambrook

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