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1. Make competent cells of an FRT-flanked cassette integrant isolate cured of pKD46. Use single-copy antibiotic concentrations for the integration cassette (or none at all, if long recovery was used during cassette integration).
   • The TSS method or even quicker, lower efficiency chemical competent cell prep methods are fine to use, such as colony-resuspension transformation, wherein a couple colonies are simply resuspended in TSS or CCMB to make cells competent enough for purified plasmid transformation.
2. Transform cells with pE-Flp [P_con-FLP ori_pSC101ts Amp^R] with recovery at 30°C. Plate Streak on LB amp⁵⁰ agar (½ normal amp conc.) and grow at 37°C 30°C to obtain colonies. Though typically not needed, you may test for loss of the resistance marker (and plasmid) as in Step 7, but with colonies tested for individual sensitivity to ampicillin (to ensure loss of pE-FLP)  and and to antibiotic antibiotic _{int cassette} (to ensure cassette excision), reserving some colony cells for preparing a frozen stock.
   • Strong, constitutive expression of Flp from pE-FLP should result in 100% resistance cassette excision without a culturing step at 30°C for prolonged plasmid maintenance.
   • Consider growing the next liquid culture at 42–43°C to further ensure curing of temp-sensitive replicon of pE-Flp. Such culture can be used to prepare a frozen stock in 20% glycerol.
   • If using the classical but inferior pCP20 [λ.p_R-FLP cI857 ori_pSC101ts Amp^R Chl^R], transform a cassette integrant with pCP20, selecting on LB amp agar at 30°C. To induce FLP expression expression and subsequent pCP20 curing, grow a pCP20-transformed colony in LB (no antibiotic) at 37° for a few hours, and replate on LB agar (no antibiotic) growing at 42°C. Grow 4–16 8–16 colonies at 37–42°C in LB, expecting <5% of colonies to have successfully excised resistance markers. Test individual colonies for loss of all resistances via plating ±antibiotics.
3. Optionally confirm integration of desired sequence by appropriate genotyping PCRs, using a fraction of resuspensions of colonies in 10 µL PBS/LB.
   1. Use PCR primers targeting the genomic context flanking the integration homologies (not inside the integration homologies) to test for successful "Flp"ing out of integration cassette, leaving a single FRT. The PCR should use an extension time long enough to make the product that would include the full integration cassette. The original strain with the integration cassette before pE-Flp treatment can be used as a positive control of the PCR detecting the cassette.
   2. You may additionally use PCR primers targeted inside the integration cassette (generally inside the resistance marker) to ensure the cassette is indeed no longer in the strain at any locus, possibly silenced, mutated, or misintegrated elsewhere.

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