Co-transfection of two plasmids - Need the protocol for lipofectamine 2000 based transfection (Nov/01/2009 )
I am trying to co-transfect (transient trasnfection) two different plasmids in to HEK-293T cells. One plasmid contains miRNA precursor and the other, 3'UTR target. Can any one briefly provide the protocol? Particularly, the amount of plasmids and lipofectamine are critical I guess.
Thanks in advance.
Go here, type in lipofectamine, choose reagent variant, download product sheet, read.
bob1 on Nov 1 2009, 04:38 PM said:
anyone else with useful suggestions please?
The protocol doesn't differ in any way! You will need to titrate the appropriate amount of each plasmid to add, but the protocol is identical.
bob1 on Nov 3 2009, 03:18 AM said:
How would I titrate the appropriate amount of each plasmid....?? I´m not asking for a protocol, just need to know the way of thinking or experimental setup and goal....
thanks in advance
Set them up at different ratios of the two plasmids e.g. a:b = 3:1, 2:1, 1:1, 1:2, 1:3, keeping the total amount (ug, copies, whatever) of plasmid the same in all treatments. For example you might want to transfect a total of 4 ug of plasmid which would give you 3:1; a=3 ug b=1 ug, 2:1; a=2.67ug, b=1.33 ug... etc.