Lipofectamine Plasmid Transfection: Difference between revisions
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[[Category: Transfection]] | [[Category: Transfection]] | ||
Materials | ==Materials== | ||
*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) | ||
==Protocol (6 well dish)== | |||
#Plate cells the day before so that they are at 90-95% confluence in 2 mL media without antibiotics, or alternatively change media before transfection to OptiMEM | |||
#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 re-feed cells with normal media. | |||
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] | ||
Revision as of 17:57, 12 August 2009
Materials
- Cells in a 6 well dish, plated and at >90% confluence
- Lipofectamine 2000 (Invitrogen cat# 11668-019 for 1.5mL)
Protocol (6 well dish)
- Plate cells the day before so that they are at 90-95% confluence in 2 mL media without antibiotics, or alternatively change media before transfection to OptiMEM
- 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 re-feed cells with normal media.
Protocol adapted from Product Manual