I was doing transfection of N2A cells in 15 cm dishes with Lipofectamine 3000 (46 ug plasmid/dish). I found a strange phenomenon that I never noticed before. When I add the plasmid to Opti-MEM medium with P3000 reagent, I saw white precipitate immdediately. I tried to pipette it, but still can not make it dissolved completely. I have done the transfection several times, but never saw it before. Does anyone know what the problem may be?
Thanks.
I can't say whether it impact the transfection efficiency yet. I'm going to collect the cells tomorrow. But I felt the precipitate looked like plasmid?
Try to reduce the plasmid amount and to respect the standard protocol ratios.
It happen if you will add Lipofectamine 3000 first followed by DNA/RNA. To avoid the precipitation you need to add first DNA/RNA followed by Lipofectamine 3000 . It will definitely work.
All the best.
Really? I did add the p3000 first. I will try to change the order next time. Thanks.
I tried the transfection again. First I warmed the Opti-MEM well, and then prepared the plasmid as Dhiraj suggested. That is adding the DNA first and then the P3000. It worked well. I did not see any precipitate. Thanks for all your answers.
I found the same problem, I mixed DNA with OPTI-MEM first ,then added P3000. Will see the transfection result though~
Yijun Zhang , definitely it will highly reduce the transfection efficiency due to precipitation. Please try the way Yinghui Li has mentioned.
I saw this as well but once I added plasmid DNA (2ug) to OPTI-MEM, followed by P3000 it resolved the issue of the precipitate.
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