After the incubation, bacterial mutation was selected by observing
“white” bacterial colonies under natural light. “White” here means
the original color of the E. coli without the gene transformed or
with the transformed DNA but damaged, whereas the normal DNA
transformed bacteria would express blue color because of the presence
of the lacZ gene. But, due to ampicillin added in the culture medium
and the ampicillin resistance of the plasmid DNA, original
bacterial cells without transformed DNA would be killed and thus,
in the final medium, white colonies, if any, should be only mutants.
White colonies were streaked in 5 generations on plates again to
check for their purities and stability of the phenotype with PCR
(polymerase chain reaction) used for selection. The number of the
white colonies was counted compared with the total number of the
tested bacterial colonies, normally thousands, for calculation of the
mutation frequency.