subsequently cloned into puc 18 using the sure clone ligation kit pharmacia southern blot analysis southern hybridization experiments were performed to construct genomic maps to find appropriate dna fragments carrying the phytase gene genomic dna 3 u03bcg was digested with the different restriction enzymes as indicated in the legends of the figures and electrophoresed on a 0 75 agarose gel the transfer to zeta probe blotting membranes bio rad was done as described in southern e m detection of specific sequences among dna fragments separated by gel electrophoresis j mol biol 98 503 1975 prehybridization and hybridization was in 7 sds 1 bsa fraction v boehringer 0 5m na 2 hpo 4 ph 7 2 at 65 u00b0 c probes derived from pcr products of the respective phytase genes see fig1 were labeled with u03b1 32 p dgtp amersham by random priming according to sambrook j fritsch e f maniatis t molecular cloning a laboratory manual second edition ed 1989 cold spring harbor n y cold spring harbor laboratory press u s a and used in the hybridization experiments after hybridization the membranes were washed twice for 5 minutes in 2 u00d7 ssc 1 sds at room temperature and twice for 15 minutes in 0 1 ssc 0 1 sds at 65 u00b0 c before exposure o n on kodak x omat ar film library construction prior to the partial library construction southern.