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Co-transfection of two plasmids - Need the protocol for lipofectamine 2000 based transfection (Nov/01/2009 )

Hi,

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.
:)

-cell_man-

Go here, type in lipofectamine, choose reagent variant, download product sheet, read.

-bob1-

bob1 on Nov 1 2009, 04:38 PM said:

Go here, type in lipofectamine, choose reagent variant, download product sheet, read.

:D wow...thanks...what makes you think I haven't already tried it...the manual describes a the protocol for 2 plasmids and siRNA transfection and not 2 plasmids.
anyone else with useful suggestions please?

-cell_man-

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-

bob1 on Nov 3 2009, 03:18 AM said:

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.


How would I titrate the appropriate amount of each plasmid....?? Im not asking for a protocol, just need to know the way of thinking or experimental setup and goal....
thanks in advance :(

-Sonchita-

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.

-bob1-