internal control - (Sep/18/2009 )

Hi,

I am transfecting (With FUGENE) my cells with metluciferase(secrets to the media) and betagalactosidase as internal standard.The luciferae promoter is CMV and betagal`s promoter is SV40.
To optimize the optimum amount of DNA to be added to each cell line I transfected cells with fixed amount of betagal and different concentrations of pCMVMetluciferase (from 0ng to 500ng) I also included the pCDN3 to have the fixed quantity of DNA in every condition. when I measured th luciferase activity it showed that after 200ng it goes linear but for the betagal activity I got some strange results; with the increasing amount of luciferase concentration the betagal activity decreased.Now it is possible that there is a competetion between promoters for transcriptional factors but I also encountred another strange thing I saw that in HT-29 cells the betagalactosidase activity in every situation is fixed and there is no difference.
Now I am wondering can anybody tell me how can I confirm that its cos of low transfection efficiency or other factors involved.I thought about a piece of DNA which is labled and it is possible to chek it with microscope but I am not sure that is there such a DNA or not.If somebody have any idea I would appreciate it .

There are fluorophore labeled DNAs available, but they are expensive. It would probably also work to use a GFP plasmid as a transfection control. It is quite possible that your B-gal plasmid is non-functional in the HT-29 cells. As you plasmids are running off different promoters the B-gal is not an ideal control!

bob1 on Sep 21 2009, 03:07 AM said:

Dear bob1
Thanks for your advice, I have already tried the GFP and it showed that the cells are transfected with FUGNE even better than LONZA machine and the NEON machine from invitrogene now I want to compare the different cell lines transfection efficiency so I need a piece of labled DNA to transfect and see the transfection efficiency directly, not waiting for expresion the other factors also would be involved.Now my problem is this:in COS-7 and SW-620 cells with the increasing concentration of luciferase the betagal activity goes down and down(even its concentration is always fixed) but in HT-29 cells this phenomena is not seen, and the betagal activity as it is excepted is fixed, now my hypothesis is that because these cells (HT-29) cells are difficult to transfect in comparison to the the other two ones therefore there is no such a big amount of luciferase activity to suppress the betagal activity (it's luciferase activity is 100 times less than SW-620 cells) and that's why I see the fixed activity of betagal. one more thing: the flurophore labeled DNAs are available from which company?for my project the money doesn't matter.

Molecular probes (Invitrogen) has stuff for doing labeling DNA. I am sure you can buy pre-labeled ones as well.

bob1 on Sep 22 2009, 02:49 AM said:

Dear bob

Thanks again,I also found a kit from MIRUS company which let you to lable your own plasmid and then transfect it.