With the ice nucleating recombinant identified, amplify the ice gene with techniques such as polymerase chain reactions (PCR). Create mutant clones of the ice gene through the introduction of mutagenic agents such as UV radiation to inactivate the ice gene, creating the "ice-minus" gene. Repeat previous steps (insert gene into plasmid, transform E. coli, identify recombinants) with the newly created mutant clones to identify the bacteria with the ice-minus gene.