Haynes:TransfectionPlasmid Lipo: Difference between revisions
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'''Mammalian Cell Transfection - Lipofectamine Reagent'''<br> | '''Mammalian Cell Transfection - Lipofectamine LTX Reagent'''<br> | ||
Karmella Haynes, 2012 | Karmella Haynes, 2012 | ||
</div> | </div> | ||
<div style="width: 800px"> | <div style="width: 800px"> | ||
'''Materials''' | '''Materials''' | ||
* Lipofectamine | * Lipofectamine LTX | ||
* Opti-MEM I Reduced serum medium | * Opti-MEM I Reduced serum medium | ||
* Antibiotic-free growth medium (no pen/strep or other antibiotics) | * Antibiotic-free growth medium (no pen/strep or other antibiotics) | ||
* Cells | |||
'''6- | |||
'''6-WELL FORMAT'''<br> | |||
'''1 day before transfection:'''<br> | '''1 day before transfection:'''<br> | ||
# Seed cultures in 6-well plates at ~2. | # Seed cultures in 6-well plates at ~2.5x10<suP>5</sup> so that cells will be ~5.0x10<sup>5</sup> at the time of transfection. Use 1.0 mL antibiotic-free growth medium (e.g., 10% FBS in McCoy’s 5A for, no pen/strep) per well. | ||
'''Transfection:'''<br> | '''Transfection:'''<br> | ||
# Warm plasmid DNA, Lipofectamine | # Warm plasmid DNA, Lipofectamine LTX, PLUS reagent, and Opti-MEM I Reduced Serum Medium to room temperature in the biosafety cabinet. | ||
# Prepare DNA-Lipofectamine complexes ( | # Prepare DNA-Lipofectamine complexes (600 μl per sample) as follows: | ||
## Label sterile microfuge ml tubes. | ## Label sterile microfuge (1.5 ml) tubes. | ||
## | ## For each transfection, bring '''2.0 μg plasmid DNA''' up to 20 μL total volume with sterile DNase-free water. Take the DNA samples to the biosafety cabinet. | ||
## | ## Add '''570 μL Opti-MEM''' to each 20 μL DNA sample. | ||
## Add | ## Add '''2.5 μL PLUS reagent''' to each DNA+Opti-MEM sample. Incubate for 5 minutes at room temperature. | ||
## Add '''7.5 μL Lipofectamine LTX''' to each DNA+Opti-MEM+PLUS reagent sample. Incubate for 30 minutes at room temperature. | |||
# Incubate cells at 37°C in a | # Add DNA+Opti-MEM+PLUS reagent+Lipofectamine mixture '''drop-wise''' to each well of cells. | ||
# Incubate cells at 37°C in a CO<sub>2</sub> incubator | |||
# (Optional) To reduce toxicity, after 5-6 hours wash cells once with warm 1xPBS to remove excess DNA-Lipo complexes. Add fresh antibiotic-free growth medium. | |||
Transgene expression should be detectable after 24 hours. | |||
<br><br> | <br><br> | ||
''For stable cells lines:'' Passage cells at a 1:10 (or higher) dilution into fresh growth medium 24 hours after transfection. Add selective medium the following day. Grow until isolated colonies appear. Transfer single colonies into 24-well plates. Expand the cells for further testing. | ''For stable cells lines:'' Passage cells at a 1:10 (or higher) dilution into fresh growth medium 24 hours after transfection. Add selective medium the following day. Grow until isolated colonies appear. Transfer single colonies into 24-well plates. Expand the cells for further testing. | ||
Revision as of 12:54, 7 January 2013
Mammalian Cell Transfection - Lipofectamine LTX Reagent
Karmella Haynes, 2012
Materials
- Lipofectamine LTX
- Opti-MEM I Reduced serum medium
- Antibiotic-free growth medium (no pen/strep or other antibiotics)
- Cells
6-WELL FORMAT
1 day before transfection:
- Seed cultures in 6-well plates at ~2.5x105 so that cells will be ~5.0x105 at the time of transfection. Use 1.0 mL antibiotic-free growth medium (e.g., 10% FBS in McCoy’s 5A for, no pen/strep) per well.
Transfection:
- Warm plasmid DNA, Lipofectamine LTX, PLUS reagent, and Opti-MEM I Reduced Serum Medium to room temperature in the biosafety cabinet.
- Prepare DNA-Lipofectamine complexes (600 μl per sample) as follows:
- Label sterile microfuge (1.5 ml) tubes.
- For each transfection, bring 2.0 μg plasmid DNA up to 20 μL total volume with sterile DNase-free water. Take the DNA samples to the biosafety cabinet.
- Add 570 μL Opti-MEM to each 20 μL DNA sample.
- Add 2.5 μL PLUS reagent to each DNA+Opti-MEM sample. Incubate for 5 minutes at room temperature.
- Add 7.5 μL Lipofectamine LTX to each DNA+Opti-MEM+PLUS reagent sample. Incubate for 30 minutes at room temperature.
- Add DNA+Opti-MEM+PLUS reagent+Lipofectamine mixture drop-wise to each well of cells.
- Incubate cells at 37°C in a CO2 incubator
- (Optional) To reduce toxicity, after 5-6 hours wash cells once with warm 1xPBS to remove excess DNA-Lipo complexes. Add fresh antibiotic-free growth medium.
Transgene expression should be detectable after 24 hours.
For stable cells lines: Passage cells at a 1:10 (or higher) dilution into fresh growth medium 24 hours after transfection. Add selective medium the following day. Grow until isolated colonies appear. Transfer single colonies into 24-well plates. Expand the cells for further testing.
