plasmid transfection

[Notch]

Hello! Hope this is the right place to post this...

I'm working on a project with mouse stem cells, mainly 46C, and one of the experiments seems to be giving weird results.

The objective is to transfect the cells with a series of plasmids, in order to run a luciferase assay. Two of the plasmids are Renilla and Hes1.
Renilla would be constitutively expressed in the cells, but, in this case, one of the two plasmids is making the cells sick (I've repeated it 3 times and have similar results).
Transfection lasts 6 hours, after which I change media.

What I'm asking is this: is it possible to transfect the cells with one plasmid, let it work for 6 hours or so, and then add the second one?

[clokke76]

Hello Notch,

i have run plenty of dual luciferase assays and published twice using them.
i really dont think it would make a huge difference, however the editors when you publish may not like it.

Did you find which plasmid is making the cells sick?
I found that renilla is a low replication plasmid (usually used to normalize), especially as it has an SV40 promoter.

Check your promoters of your constructs and what cell line you are using. Some cell lines have much higher expression (potential toxicity than others).

Also, have you tried using a transfection reagent eg FUGENE6 that doesnt require you to starve your cells?

please let me know what you discovered
thanks

[Notch]

Speedy reply! Thank you!

Well, some more details: I'm actually an undergrad student, I'm currently doing an honours project so the chances of getting this published are slim to none. Therefore I'm not that worried about editors and such
I am however limited in terms of what materials I can use, since it is at the expense of my supervisor.

So far results are good, the Hes1 transfection protocol is a fairly new experiment that I've been doing mainly to support the results I have from previous transfections in which I've used Notch as a plasmid.

My guess is that there is something in the Hes1 plasmid which the cells don't like. It's a guess because for all my previous dual luciferases I've used the same Renilla SV40 and everything went smoothly.

I will be repeating the experiment with E14tga as well.


Have never heard of FUGENE6, but will definetely look into it! (still plenty to learn obviously!)

Thanks again for the help, will let you know how it goes!

[admin]

Hello Notch,
welcome to the forums

how much DNA are you using in the transfection? Sometimes too much is a bad thing.

Yes Fugene is great I used it too without changing the media. Some cells cant take serum free media for long.

cheers

[Notch]

Thanks for the welcome!

I did decreased the amount of DNA I transfected a few weeks ago, from 0.25ug to 0.1ug, but it didn't change anything...