Hello Secil,

What I would recommend is to dilute your transfection reagent first into DMEM and let it sit for 15-20 minutes then add recommended concentration of DNA and don't let your complex to sit too long in the medium. Precipitation is not very common but if it does happen mix it thoroughly before putting into the cells.

Has anyone used these plasmids successfully for expression before or its your first atempt to do so?

Kolay gelsin

Thanks for the answer Ahmad Jawad Sabir. The precipitation has been occured before mixing with transfection reagent, when diluting plasmid with optimem/DMEM and P3000. Pipetting or vortex didn't help at all. I used these plasmids before many times, there was no such problem.

Thanks Rachel Gad! 1 ug DNA is recommended for 24well-plate. For some other reasons I'm using 12well-plate and 2 ug DNA accordingly. It worked well until now. For dilution I've tried both optimem and serum-antibiotic free DMEM when preparing the transfection mix and precipitation came up in both situation. Otherwise after plasmid isolation I'm using ultra pure nuclease free water for resuspension and further dilution. I've checked the bands recently, they were fine. I proceeded despite of the pellet, only a few cells were transfected. However, yesterday I decided to exclude P3000 and transfection efficiency is over than %50. I think I'll go without P3000 until problem will be solved.