915
edits
Changes
Wrote protocol from ethan's trials
[[Category: Cell Culture ]]
[[Category: Transfection]]
[[Category: Muscle Cells ]]
[[Category: C2C12 Cells ]]
==Materials==
*Cells in a 6 well dish, plated and at >90% confluence
*Lipofectamine 2000 (Invitrogen cat# 11668-019 for 1.5mL)
*OptiMEM
==Required Amounts==
*Calculate DNA to transfect. Start with 4 ug per 6 well
*Lipofectamine(in uL) = DNA(in ug) * 2.5
*OptiMEM(in uL, equal volume for DNA and Lipofectamine) = Lipofectamine(in uL) * 25
==Protocol (6 well dish)==
#Plate cells and grow to ~70% confluence in 2 mL media without antibiotics (DMEM with 10% FBS, no PSG; use 1 mL for 12 well plate, and use half the amount of DNA)
#For each well prepare one tube for DNA and one tube for Lipofectamine:
##OptiMEM plus required total amount of DNA.
##OptiMEM plus required total amount of Lipofectamine.
##Incubate ~5 minutes at room temperature.
##Combine the equal volumes of OptiMEM/DNA and OptiMEM/Lipofectamine.
##Incubate ~20 min at room temperature.
#Add DNA/Lipofectamine complexes (~500 uL) to the 2 mL of media on the cells
#Gently rock plate to mix
#After ~4h aspirate media and re-feed cells with normal media. (It is usually possible to leave overnight).
#Examine the cells the next day.
Protocol adapted from [http://tools.invitrogen.com/content/sfs/manuals/lipofectamine2000_man.pdf Product Manual]
[[Category: Transfection]]
[[Category: Muscle Cells ]]
[[Category: C2C12 Cells ]]
==Materials==
*Cells in a 6 well dish, plated and at >90% confluence
*Lipofectamine 2000 (Invitrogen cat# 11668-019 for 1.5mL)
*OptiMEM
==Required Amounts==
*Calculate DNA to transfect. Start with 4 ug per 6 well
*Lipofectamine(in uL) = DNA(in ug) * 2.5
*OptiMEM(in uL, equal volume for DNA and Lipofectamine) = Lipofectamine(in uL) * 25
==Protocol (6 well dish)==
#Plate cells and grow to ~70% confluence in 2 mL media without antibiotics (DMEM with 10% FBS, no PSG; use 1 mL for 12 well plate, and use half the amount of DNA)
#For each well prepare one tube for DNA and one tube for Lipofectamine:
##OptiMEM plus required total amount of DNA.
##OptiMEM plus required total amount of Lipofectamine.
##Incubate ~5 minutes at room temperature.
##Combine the equal volumes of OptiMEM/DNA and OptiMEM/Lipofectamine.
##Incubate ~20 min at room temperature.
#Add DNA/Lipofectamine complexes (~500 uL) to the 2 mL of media on the cells
#Gently rock plate to mix
#After ~4h aspirate media and re-feed cells with normal media. (It is usually possible to leave overnight).
#Examine the cells the next day.
Protocol adapted from [http://tools.invitrogen.com/content/sfs/manuals/lipofectamine2000_man.pdf Product Manual]