I did transfection to 90% confluent HEK-292 cell line with pNL4-3 DNA and p-Indie-C1 DNA using lipofectamine 2000 in 6 well culture plate. My experiment recipe is as:
1. 5 ul Lipo +250 uL DMEM each well, incubated for 5 min before mixing to DNA
2. 2ug DNA + 250 uL DMEM
2. mixing of Lipo and DNA
4. Upto that time cells were starved, complete media was removed and added serum free media to each well.
5. 500 uL mixture of Lipo and DNA was added to each well containing 250 uL serum free media.
The result showed good transfection with pNL4-3 but pIndie-C1 showed floating and not adherent cells of HEK and cells are lost..after aspiration of media..
Is there a valid protocol for transfection of pIndie-C1 to HEK cells?
Firstly, what concentration are your plasmids at? It sounds like the amount of pIndie-C1 you are adding is toxic to your cells. Before transfection experiments I always do a titration to determine the best combination of amount of Lipofectamine & amount of DNA, to the highest amount that does not kill the cells.
I used the 2 ug DNA for each well of six well plate. (The concentration of pIndie-C1 is 0.6 ug/uL.
What did you use to purify pIndie-C1? High LPS/endotoxin concentrations may also ruin your transfection...
Alejandro,
I prepared the pIndie-C1 plasmid through Gen-JET Maxipreparation protocol.. With same procedure, the result of pNL4-3 was good.
You have several possibilities to explain the toxic effect:
-Too much DNA and thus lipofectamine, lower the amount of DNA is possible
- Cells are too confluent (lower density to 70%). make sure that your cells are healthy and mycoplasma free (combination of contaminants with some gene might lead to high toxicity)
- Expression of pIndie-C1 might lead to the toxicity
- Be sure to use endotoxin free plasmid preparation kit
Finally you can try other transfection reagent: DreamFect Gold is a good one.
I hope this can help
Thankx Olivier.. As my HEK cell lines got contaminated. Can I use HELA cell line? Regards
Thank you Oliver..
Do you think that the trasfection efficiency of HEK cells are better that HELA cells?
What are the differences of transfection in these cell lines.
Regards
Hi Bivek, usually efficiency is supposed to be better with HEK but you can achieve up to 80% efficiency on Hela with some reagent such as dreamfect gold. Here a two recent references
http://www.ncbi.nlm.nih.gov/pubmed/23665581
http://www.ncbi.nlm.nih.gov/pubmed/23508956
Thank you Olivier..
I am sorry that I am not able to get full article you sent. And in abstract I didn't get. Could you please help me.
Regards
If you give me your email address I can send you the articles
Best Regards
Thank you Olivier.
My gmail id is: [email protected]
I am pleased to get help from you.
Regards
I am not getting proper transfectiion result with p-INDIEC-1 using 2ug DNA per well. I have collected the soup after 24 hours and then again after 2 hours. What can I do in this case. I am getting very faint band after western blotting. I need to increase the transfection and so the viral titre.
Suggestions are highly appreciated.
Regards.
I have checked the transfection efficiency using fluorescence microscopy using GFP tagged DNA and also using FACS analysis using FITC. I am getting low transfection efficiency. I suspect that the quality of DNA may be not as good as needed. Please suggest me. Thank you.
Bivek
Maybe your DNA construct is not pure enough, I suggest using endotoxin free purification kit. I hope this can help
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