oligo 1 50 000 dil of sybr u00ae green 0 25 mg ml bsa 1 unit taq polymerase and h 2 o to 20 u03bcl pcr buffer ii is available in 10 u00d7 concentration from perkin elmer norwalk conn in 1 u00d7 concentration it contains 10 mm tris ph 8 3 and 50 mm kcl sybr u00ae green molecular probes eugene oreg is a dye which fluoresces when bound to double stranded dna as double stranded pcr product is produced during amplification the fluorescence from sybr u00ae green increases to each 20 u03bcl aliquot of amplification mixture 2 u03bcl of template rt was added and amplification was carried out according to standard protocols cells were seeded into 96 well plates at a density of 5000 cells per well for a 4 day proliferation assay 5 independent 96 well plates were prepared one for each day after overnight incubation cells were transfected using the procedure described above on each day of the proliferation assay all medium was removed from one plate and frozen at u2212 70 u00b0 c on day four all plates were developed with the quantos u2122 assay kit stratagene la jolla calif which determines the amount of dna and thus the number of cells in each well cells were seeded in 35 mm dishes at 35000.