• We never use endotoxin free kits just home made buffers and more than 10 years we never had problems with transfection in Hek293. Can be a possibility that IL12 starts a signal leading to cell death, a very naive idea?

Thanks Umair.

I would like to update my downstream data. I tried six constructs that were prepared by mini-prep, namely A, Bn, C1, C2n, Be and C2e, where n and e indicate the native and engineered versions of two genes.

I used nearly 1 ug (~1.5 uL in TE buffer) of plasmids for transfection using the x tremegene 9 kit. After 24 h, cells remained healthy and it implies that endotoxins present in the mini-prep is either negligible or HEK293T cells are much tolerant against the small amount of endotoxins.

Then I processed the media (containing secreted protein) and the cells (containing non-secreted proteins) and performed the SDS-PAGE and Western blot (Refer the Figure).

Based on results, we can guess that engineered version of B and C2 are not secreted.

As Umair indicated for final experiments, it is better to use the transfection-grade midi kits. So I proceed with A, Bn, C1 and C2n to upscale to get much pure plasmids.

Using mini-prep kit in a preliminary experiment would help us avoiding unnecessary waste of time and resources in a clone/construct which is not productive (such as Be and C2e).

I hope this would be helpful for someone.

Thanks Molnár.

There was no failure for me too. But, before my attempt, I was curious to know why one cannot use the mini-prep kits that yield only a few micrograms of DNA (possibly with endotoxin as contaminants), rather than using midi-kits. I hope my updates made it clear.

Thanks again.

Dear Navaneethaiyer,

It does not matter what kit (mini, midi, maxi etc) you use it is basically only upscaling the isolation. The basic kits do not contain the removal of endotoxin, which is not necessary if you use your DNA in normal cell culture. The endotoxin free kit may be required if you work will stem cells or in vivo animal or human studies. The size of your isolation mini versus midi depends how much DNA you will use and indeed when we were just checking couple of wells we used DNA from mini preps. Worked same as from maxi preps. I would really concentrate and read about what IL12 does generally to cells and how close is human and fish IL12 (I feel that there is rather high conservation).

Dear Molnár,

Thank you very much for the valuable comments. I clearly understood the idea and the context at which the transfection-grade kits are necessary.

Thanks again.

Hi Zhen,

Precisely, there was no difference. In my opinion, you can use mini-preped plasmid in your preliminary assay to check whether the system works well. Later on, you can use highly pure transfection-grade plasmid in your final experiments.