No resistant colonies were obtained in control experiments, in which the plasmid DNA was omitted. Very high variability in number of transformed colonies per plate was observed, indicating a central role of cells' handling and recovery, such as: cultivation, plating, re-plating and incubation conditions during the recovery period on the apparent transformation efficiency. Conditions applied were as described in Materials and methods. Testing bombardment with different rupture disc calibers or bombardment distances yielded either equal or worse transformation frequencies. The best relative transformation frequency was achieved with two days old cells plating 3 × 106 per plate. Transformed colonies were maintained through successive platings for more than 33 months and stable insertion of the mutated gene was verified by extended periods of cultivation both in the absence or presence of selective pressure, and subsequent PCR amplification and sequencing of the pds.
No resistant colonies were obtained in control experiments, in which the plasmid DNA was omitted. Very high variability in number of transformed colonies per plate was observed, indicating a central role of cells' handling and recovery, such as: cultivation, plating, re-plating and incubation conditions during the recovery period on the apparent transformation efficiency. Conditions applied were as described in Materials and methods. Testing bombardment with different rupture disc calibers or bombardment distances yielded either equal or worse transformation frequencies. The best relative transformation frequency was achieved with two days old cells plating 3 × 106 per plate. Transformed colonies were maintained through successive platings for more than 33 months and stable insertion of the mutated gene was verified by extended periods of cultivation both in the absence or presence of selective pressure, and subsequent PCR amplification and sequencing of the pds.
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