Please Help! Transfection: dropwise addition of lipid -dna complexes
Posted 05 December 2009 - 02:43 PM
I think I may be trying to hard to mix the optimem+complexes with the serum media the cells are in, and that the force(although I try to be gentle) resulting from my thoroughness is enough to cause cells to clump and detatch from plate.
I am concerned because my plates were ~70% confluent this morning, but after I changed media and added the lipid-dna complexes, it seems like only 50%(or fewer) cells are attached. Is there anything I can do to fix this before I harvest tomorrow morning?
Thanks in advance.
Posted 06 December 2009 - 12:07 PM
Posted 06 December 2009 - 03:00 PM
Posted 06 December 2009 - 04:42 PM
Also, is the nucleic acid in the media responsible for cell-cell clumping? If so, how could I prevent it from happening when doing a Reverse transfection.
Thanks for the reply
Posted 07 December 2009 - 02:52 PM
The clumping isn't because of the DNA of the transfection reagent, It is much more likely that you over trypsinised the cells or that there is a vibration in the incubator which will cause the cells to settle in concentric rings (in dishes /6 well plates - you won't be able to detect it in 24 well plates) or lines in flasks. This can be intermittent and is usually more obvious with cells that are taking a while to settle (like yours) as they have more time to get shuffled into the waveforms.
Also don't believe the optimem stuff invitrogen goes on about. Just use your normal medium (without FBS), it will be fine. I have had less toxicity with Fugene 6 in the past too, if that is any help.
Posted 07 December 2009 - 03:25 PM