I'm going to be doing some transfections of MCF-7 and MDA-MB-231 cells, with a plasmid containg CAT, and my gene of interest. We have lipofectamine 2000, and I'm just going through the protocol that is suggested by said manufacturer...
I will be using a 12 well plate, so, according to protocol, I should use 1.6ug DNA diluted in 50uL .... blah blah blah.... but in some papers i've been reading, they've used in a 24 well plate 12 ug of CAT plasmid. Help? This is a huge difference, and i'm not sure if a, this is a typo in the paper (maybe 1.2ug?).
Also, according to manufacturer, transfection should take 24-48 hours.... other papers say 6 hours. Which is best a little time, or a lot?
And another thing, someone suggested that the cells should have a small treatment in glycerol after the transfection medium is removed, for 10 minutes or so. I've never heard of this before, and can't find anything relating to this? Suggestions really appreciated.
help me please, limited time frame to do this work in. help me, i've fallen over and can't get up.
/ how do you tell if transfection has taken place? facs or something? have no idea on this.
Edited by vetticus3, 16 March 2005 - 07:50 PM.