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Figure 2 | BMC Microbiology

Figure 2

From: A recombinase system facilitates cloning of expression cassettes in the ciliate Tetrahymena thermophila

Figure 2

Analysis of the Cre-recombinase reaction with novel donor and acceptor plamids. Results of the Cre-recombinase reactions using pKOIX, pAX, and pDL-MSP119, respectively. Left column: Analysis of recombinant pAX-MSP119 clones. Top: The restriction analysis of recombinant pAX clones with XhoI and SacI and are shown. The analyzed positive clones (1–6) have an 8.6 kb backbone and a 1.2 kb insert. The vector backbone without the insert (control B) and the donor plasmid insert (MSP119) are shown in lanes B and I. Bottom: The diagnostic PCR confirms that all analyzed clones are positive, because the amplification of the specific 360 bp fragment is only possible in recombinant clones. The acceptor control (A) is negative and the fragment in the donor control (D) is an unspecific artefact (200 bp). Right column: The analogous approach as shown in the left column with the acceptor plasmid pKOIX instead of pAX. The analyzed positive clones carry a 7.9 kb backbone and the 1.2 kb insert. The additional band at 1.0 kb is due to a SacI site in the Cm resistance. Lanes B and I show the used backbone (pKOIX) and the 1.2 kb insert (MSP119). The diagnostic PCR leads to the specific 360 bp fragment like in the pAX-MSP119 approach. The 200 bp fragment in the donor control, (D) is an unspecific artefact. The acceptor control (A) is negative.

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