not successful with transfection - (Nov/05/2014 )

Hi

Iam using a stable clone which expresses my protein of interest, with G148 resistance and I use 500ug/ml of G418 in the medium which is DMEM. Now I want to transfect with another plasmid having a cell cycle probe. Since this dosent have any selectable marker, Iam using puromycin which is in linear form.

First I plate the stable clone without G418 in the medium and the next day, I use 1ug of DNA of the plasmid having cell cycle probe and 100ng of puromycin. I transfect with x treme gene 9, change the medium after 6 hrs and allow the cell to express for 48 hrs.

After two days, I change the medium which contains both G418 and puromycin (2ug/ml, conc determined by the kill curve). The next day all the cells died, which means that my experiment failed. what is want to know is

1. why is this happening

2. what is the rate of success when doing a experiment like this starting with a stable clone instead of using a normal cell line.

3. what should I do to get this successful.

please help

I don't understand why you are using puromycin when there is no selectable marker... puro will kill all your cells unless you have a resistance gene present.

Iam transfecting puromycin in 1/10 the conc of DNA

But you still don't have a puromycin resistance gene, it will kill your cells...

That is why I wanted to know how to do the transfection successfully. I have done it more than 3 times but without any success. Will transfection in a stable clone be more successful than in a cell line. Because I was wondering since the stable clone is expressing a protein already, will it be under more stress to take another plasmid and the added puromycin

You can't make a stable line by transfecting a cell with a plasmid that doesn't have a selectable marker. In your case the best you could do is a transient transfection. Adding puromycin isn't going to help you select as you don't have the resistance marker. The stable clone which you have already shouldn't be under any more stress than a normal cell line unless it is a toxic protein of some sort or expressed at very high levels.