size of any observed fragment depends on the insertion event the number of bands can be used to estimate insertion copy number u2014 each gene copy would produce a unique band on the southern blot the results of a southern blot are summarized in table 2 these data show that the bt11 transgenic lines are derived from a single insertion event containing one gene copy of the bt and pat gene sequences the dna probe fragments were isolated from the original plasmid vector pzo1502 btk sal i and sac i fragment and pat sal i fragment fresh tissue samples 30 50 mg were ground on ice in u02dc 5 volumes of extraction buffer 100 mm tris hcl ph 7 5 3 mg ml dithiothreitol and 0 3 mg ml bovine serum albumin bas fraction v the homogenate was centrifuged to clarity 12 000 u00d7 g for 5 min approximately 2 u03bcl of extract was added to the reaction mixture containing the extraction buffer plus 125 u03bcm acetyl coa and 250 u03bcm phosphinothricin the enzymatic reaction was allowed to proceed for 1 hour at 37 u00b0 c the reaction mix was then spotted onto tlc silica gel plates baker si250 pa 19c the plate was chromatographed for 2 3 hours.