pGL3 Promoter activity is higher than pGL3 Control? - (Nov/25/2007 )

Hi all

I have repeated my assay twice (n=2) where each insert is done in quadruplicates. My results show that pGL3 Promoter activity is higher than pGL3 Control after normalising with pRL TK. Is this normal? The difference between pGL3 Promoter and pGL3 Control that i'm aware of is the absence of SV40 enhancer in pGL3 Promoter. Does this mean that the SV40 enhancer is actually a repressor in 16HBE (cells i used for transfection) ???????


Thanks y'all

First, make sure that you did not mixed up the two.

16HBE is a line established from Adv-SV40 hybrid virus infection, so it should have SV40 T. plasmids with SV40 replication origin, such as pGL3 control should be able to replicate in these cells. Any abnormal morphology and cell death in these cells after transfection with promoter and control cells?

please dont multi-post the same content.

please dont multi-post the same content.

You can wash the cells after 48hr post transfetcion, measure for cell-associated protein. This gives you a rough idea how much cells remained in the well in a relative term. Or you can stain for nuclei with propidium iodide and count the cells in the wells using a fluorescence microscope with rhodamine channel. Sometimes, cells with too many copies of a plasmid will get them killed.

You may also do a transfection with a different cell type with these vectors. 293, hela, etc. Just avoid Cos 1 or 7 cells because SV40 ori-containing plasmid will replicate in these cells.

Again, verifying if you are working with the right constructs will be the first step to do. Just cut them with REs and run a gel to be sure.

I've checked my vector using RE and they are of the right size. I couldnt have mixed them twice in a row. Can it be that the vectors are not working correctly?