Hi. I'm trying to transfect CAL27 cell line (squamous cells of the tongue) with an overexpressing plasmid (size 7.7 Kbp) in different concentrations (from 50 to 1000 ng) using different volumes of TransIT Reagent by Mirus for different times, with no results (while I had no problems with siRNAs), besides partial death (same death with the empty plasmid).
Do you have any experiences with cells hard to transfect with plasmids? Do you have any suggestions for me?
(Plasmid works very good in HEK293T)
Thank you in advance for your attention and answer.
Dear Mariangela Palazzo ,
you should try a different transfection reagent. I do not see the point why a 7.7 kbp Plasmid is hard to transfect. I assume that you have used simple CaPO4 transfection for the Hek cells? Try to get Lipofectin 2000 or 3000. You might ask a colleague from a different department or your sales representative?
According to my quick literature search you might be even able to transfect them with the CaPO4 method. You should use an empty GFP coding plasmid a positive control.
Best wishes
Soenke
Hi, according to your mail, you have tested the conditions for transfection so maybe the pb comes from the promoter that drives the expression of your gene of interest (promoter that could work in HEK cells but not in your Cal27... ) you can also use a very different way to transfect like electroporation (but requires an expensive apparatus...) . are Cal27 known to be hard to transfect? also maybe set up the conditions with an easy to follow reporter gene (luciferase, eGFP... ) to be abble to screen more combinations....
Hi Sönke Weinert Didier Poncet . thank you for your answers. So far in CAL27 I have tried different types of transfection reagents (TransIT Mirus, Transfectin BioRad, Lipofectamine 2000 Invitrogen) in different ratios with my plasmid and also electroporation and none of these methods worked checking results by western blot. I have checked the efficiency of electroporation with GFP plasmid and it seems to be less than 10%.
any further suggestions?
Thank you in advance.
Dear Mariangela Palazzo ,
My be it's a problem of confluence or serum during the transfection (although I have worked with Lipofectamine in the presence of serum. Maybe you can share a little more details about your transfection method. Cell density, medium, and transfection reaction to make another suggestion for the trouble shooting.
Do you have other colleagues in the lab, who are transfecting the CAL27 cells with success? You might ask them to transfect your cells with a empty GFP construct as an control.
Best wishes Soenke
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