I would suggest transfecting 1ug of DNA into each well (6-well format). I have found that most cells don't really like getting more than 1ug and start dying the day after transfection. In my opinion, just adding the same amount of DNA to each well should be enough - I would think mass differences wouldn't be large enough to make much of a difference.

 if I understand well, you want to co-transfect 2 or more plasmids of different sizes. Then, you should not consider the number of plasmid units but only the amount of DNA.

If the protocol for your reagent propose to start with a soltuion of 10ng/ul for example, then prepare a mix to have this amount of DNA with a ratio corresponding to the size difference between your plasmids. E.g. if you have 2 plasmids, one small and one 2-fold bigger, use 2 times more of the small one.

HEK are standard cells and are very good hosts for transfection. Most of available commercial reagents can be used without optimization, and even with lower quantities as indicated in manufacturers' protocols.

As a reference, our Viromer RED works perfectly fine with 50-100ng/96-well (i.e. 250-500ng/24-well, or 1-2ug/6-well). In-house, we usually test our reagent with HEK cells at a seeding density of 2.210^4/96-well (for a read-out after 24h).

If you want to learn more about transfection optimization, please have a look to the attached file. From p9, you will find many tips (applicable to all chemical transfectants).

Good luck!

Sandra

I remember I had the same dilemma myself when comparing variants of the same protein with different length. I think the right procedure will be to use the same amount of inserts, i.e. DNA molecules and not the same amount of DNA. However, we do not know whether the expression of proteins with different length will be the same.

Is the increase or decrease in the plasmid number in the transfection concoction going to affect the plasmid number per cell in your culture? It appears that the answer is probably NO. Then you are left with the other option i.e. take a measure by the amount of DNA (which is the mass of DNA). Hope this is useful !

Best wishes