Recently I performed arabinose-induced flp-mediated neo deletion in SW105 (DH10B derivative) and tried to confirm neo deletion by PCR. PCR primers bind to 5' and 3' flanking regions of upstream and downstream frt, respectively. I confirmed the PCR product corresponding to a size to indicate neo deletion. However, using primers binding to the adjacent regions of each frt (one primer in each primers set binds to the region to be excised after ara-flp), PCR amplified the same size as seen in uninduced control. I have heard that DH10B and its derivatives can maintain only 1 copy of BAC DNA. Is it true? Is the same true in plasmid? If bacteria has multiple copy number of BAC DNA and ara-flp neo deletion could be incomplete, the incubation time for flp induction might be insufficient.
PCR is very sensitive for detecting the NEO excision after Arabinose treatment. In some cases I saw a fraction of colonies that I screened had evidence for both NEO excision and no excision. However, I always get complete NEO excision in the majority of colonies, so just go with those clear ones for further steps. It is not worth worrying about the unclear ones, as this could easily also reflect contamination in the PCR or other technical error. If you don't get any clear colonies with full excision then you have a problem worth tracking.
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