u03b2 actin egfp was released with bamhi and kpni blunted with t4 dna polymerase and cloned into hs4 u03b2 actin puro digested with bglii and ecorv five transfections were performed using this construct for each transfection 5 u00d7 10 6 pgcs were resuspended in 400 u03bcl electroporation buffer specialty media and 20 u03bcg of linearized dna was added an ed pulse 150 200v 900 u03bcf or sw 350v 8 pulses 100 u03bcsec pulses were given after transfection the cells were plated into individual 48 wells and grown for several days before selection 0 5 u03bcg ml was added a total of 5 clones were observed in 4 of the 5 transfections one clone tp103 was analyzed by southern fig1 referring to fig1 the plasmid control dna was linearized with noti an internal fragment was released by digesting the dna with kpni in both tp103 and the plasmid a fragment of the same size was released digestion of genomic dna of tp103 with ncoi mfei and sphi should reveal bands that are larger than the corresponding lanes of digested plasmid dna no band is seen in the lane of mfei digested tp103 genomic dna which may be due to the band being too large in the lanes representing the ncoi and sphi digestions fragments have been released in the tp103 genomic dna that are substantially larger than the fragments released in the plasmid dna indicating that the transgene is incorporated into the genome of the tp103 cell line first u03b2.