FLP-mediated conditional loss of an essential gene to facilitate complementation assays Page: 27
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ligated to itself for use as template in a final round of PCR. The amplification of the mutagenized
Bar gene was carried out using the primers BAR-Kpn3 and BAR-Hind5. Cycling conditions for
the second and final amplification are listed in Table 3 and Table 4, respectively. The final
amplified mutagenized Bar gene product was digested with restriction enzymes HindIII and
KpnI and ligated into the pFLP-SWITCH backbone also digested with HindIII and KpnI. The
ligated mixture was transformed into E. coli cells. Plasmid DNA was isolated and recombinant
clones were tested for the presence of the insert by a HindII/KpnI restriction digest. The
mutagenesis of the undesirable KpnI restriction site in the BAR gene was also confirmed by
sequencing. This plasmid is referred to as pFLP-SWITCH-BAR.
3.9.2 Construction of pGEM-ecSUC2-BAR
The pFLP-SWITCH backbone contained an inconvenient SacI restriction site. Therefore,
the NotI fragment from pFLP-SWITCH-BAR that excluded the inconvenient SacI site was sub
cloned into the NotI site of pGEM-T-easy cloning vector (Promega, Wisconsin, USA) creating
the plasmid pGEM-BAR (Fig. 10). The ligation product was transformed into E. coli cells and
positive clones identified by digestion with HindIII restriction endonuclease. (A SacI site in the
pGEM-T-easy backbone was mutated prior to sub cloning by digesting with SacI, treating with
T4 DNA polymerase to make the SacI digested ends blunt and religating). The AtSUC2 cDNA
under the control of its 2 kb native promoter was obtained as a BamHI/SacI fragment from the
pGEM-SUC2p/SUC2 plasmid (Ayre et al., unpublished) and cloned into BamHI/SacI digested
pGEM-BAR backbone, creating pGEM-ecSUC2-BAR. The insertion of the SUC2pSUC2
cassette into the pGEM-BAR backbone was analyzed by restriction digestion with the restriction
endonuclease KpnL The desired fragment sizes of 6.5 kb and 2.5 kb were ontained, confirming27
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Ganesan, Savita. FLP-mediated conditional loss of an essential gene to facilitate complementation assays, thesis, December 2007; Denton, Texas. (https://digital.library.unt.edu/ark:/67531/metadc5180/m1/37/: accessed July 16, 2024), University of North Texas Libraries, UNT Digital Library, https://digital.library.unt.edu; .