Lipofectamine Plasmid Transfection: Difference between revisions
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*Cells in a 6 well dish, plated and at >90% confluence | *Cells in a 6 well dish, plated and at >90% confluence | ||
*Lipofectamine 2000 (Invitrogen cat# 11668-019 for 1.5mL) | *Lipofectamine 2000 (Invitrogen cat# 11668-019 for 1.5mL) | ||
==Required Amounts== | |||
*Calculate DNA to transfect | |||
*Lipofectamine(in uL) = DNA(in ug) * 2.5 | |||
*OptiMEM(in uL, equal for DNA and Lipofectamine) = Lipofectamine(in uL) * 25 | |||
==Protocol (6 well dish)== | ==Protocol (6 well dish)== | ||
#Plate cells the day before so that they are at 90-95% confluence in 2 mL media without antibiotics. | #Plate cells the day before so that they are at 90-95% confluence in 2 mL media without antibiotics (DMEM with 10% FBS, no PSG). | ||
#For each well prepare: | #For each well prepare: | ||
##250uL OptiMEM plus 4 ug of DNA. | ##250uL OptiMEM plus 4 ug of DNA. | ||
| Line 16: | Line 21: | ||
#Add DNA/Lipofectamine complexes (~500 uL) to the 2 mL of media on the cells | #Add DNA/Lipofectamine complexes (~500 uL) to the 2 mL of media on the cells | ||
#Gently rock plate to mix | #Gently rock plate to mix | ||
#After ~4h re-feed cells with normal media. | #After ~4h aspirate media and re-feed cells with normal media. (It is usually possible to leave overnight). | ||
Protocol adapted from [http://tools.invitrogen.com/content/sfs/manuals/lipofectamine2000_man.pdf Product Manual] | Protocol adapted from [http://tools.invitrogen.com/content/sfs/manuals/lipofectamine2000_man.pdf Product Manual] | ||
Latest revision as of 21:00, 11 July 2012
Materials
- Cells in a 6 well dish, plated and at >90% confluence
- Lipofectamine 2000 (Invitrogen cat# 11668-019 for 1.5mL)
Required Amounts
- Calculate DNA to transfect
- Lipofectamine(in uL) = DNA(in ug) * 2.5
- OptiMEM(in uL, equal for DNA and Lipofectamine) = Lipofectamine(in uL) * 25
Protocol (6 well dish)
- Plate cells the day before so that they are at 90-95% confluence in 2 mL media without antibiotics (DMEM with 10% FBS, no PSG).
- For each well prepare:
- 250uL OptiMEM plus 4 ug of DNA.
- 250uL OptiMEM plus 10 uL of Lipofectamine.
- Incubate ~5 minutes at room temperature.
- Combine the two 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).
Protocol adapted from Product Manual