5 replies to this topic

[cell_man]

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.

[bob1]

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

[cell_man]

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

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

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?

[bob1]

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.

[Sonchita]

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....?? I´m not asking for a protocol, just need to know the way of thinking or experimental setup and goal....
thanks in advance

[bob1]

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.