I'm trying to do transient transfection on snu638 cell line. I'm using flag tag plasmid for transfection , initially it got worked. But if I try to repeat it it is not working I tried different transfection reagents using takara xfect. Fugene and lipofectamine.but not able to get flag expression. Cells were 70 to 90% confluent qt the time treansfection.and I used to collect lysates after 48 hrs of transfection.