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Co-transfection of two plasmids


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#1 cell_man

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Posted 01 November 2009 - 11:15 AM

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.
:)

#2 bob1

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Posted 01 November 2009 - 03:38 PM

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

#3 cell_man

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Posted 01 November 2009 - 08:16 PM

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?


#4 bob1

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Posted 02 November 2009 - 04:18 PM

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.

#5 Sonchita

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Posted 11 November 2009 - 08:47 AM

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 :(

#6 bob1

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Posted 11 November 2009 - 04:42 PM

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.




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