View source for Lipofectamine Mediated Knockdown

[[Category:  Cell Culture ]]
[[Category:  Knockdown]]

==Materials==
*Cells in a 12 well dish
*Lipofectamine 2000 (Invitrogen cat# 11668-019 for 1.5mL)

==Required Amounts==
*Calculate RNA to transfect (typically 40 pmol per well)
*Lipofectamine(in uL) = RNA(in pmoles) * 50
*OptiMEM(in uL, equal for DNA and Lipofectamine) = Lipofectamine(in uL) * 50

==Protocol (6 well dish)==
#Plate cells the day before from a confluent dish diluted to 1:12 into DMEM/FBS with no PSG
#For each well prepare, scalin up as required:
##100uL OptiMEM plus 40 pmoles of RNA.
##100uL OptiMEM plus 2 uL of Lipofectamine.
##Incubate ~5 minutes at room temperature.
##Combine the two volumes of OptiMEM/DNA and OptiMEM/Lipofectamine (should be 200uL/well).
##Incubate ~20 min at room temperature.
#Add DNA/Lipofectamine complexes (~200 uL) to the 1 mL of media on the cells
#Gently rock plate to mix
#After ~4h re-feed cells with normal media.

Protocol adapted from [http://tools.invitrogen.com/content/sfs/manuals/lipofectamine2000_man.pdf Product Manual]