). Insertion of new DNA into any of the lacZ restriction sites causes inactivation of the gene and the loss of beta-galactosidase activity. Recombinants and non-recombinants can therefore be distinguished by plating the transformed cells on ampicillin agar plates containing X-gal.
All cells growing on the medium are transformed cells because only transformed cells are ampicillin-resistant. Some colonies will be blue and some will be white (DNA, 1999). The blue colonies contain cells with functional beta-galactosidase enzymes so must have undisrupted lacZ genes and therefore are non-recombinants White colonies are composed of cells without beta-galactosidase activity, which means the restriction lacZ sites have been blocked by new DNA insertions and these are the colonies containing recombinant cells. If the cells from the blue colonies contain a small insertion, then it is obviously inserted at a different site than the lacZ insertion site, and therefore does not interfere with beta-galactosidase activity.
The lack of an insertion gene in the white cells in this example is no doubt due to the size of the viral DNA (Luz, 2004). A problem with using pBR322 and pUC8 is that these plasmids are unable to handle large DNA fragments - those greater than about 10 kb in size. If the fragment of viral DNA used was larger than this, it may have interfered with the plasmid replication system in such a way that the recombinant DNA molecules were lost from the cells.
To insert a gene for galactonolactone dehydrogenase (GLDH) from cauliflower into potato, first the DNA containing the gene for GLDH would be cut fr
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