Haynes:TransfectionPlasmid Lipo: Difference between revisions
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<- [[Haynes:Protocols | Back to Protocols]] | <- [[Haynes:Protocols | Back to Protocols]] | ||
<div style="width: 800px | <div style="width: 800px"> | ||
=Mammalian Cell Transfection - Lipofectamine LTX Reagent= | |||
Karmella Haynes, 2012 | Karmella Haynes, 2012 | ||
==Materials== | |||
* Lipofectamine LTX | * Lipofectamine LTX | ||
* Opti-MEM I Reduced serum medium | * Opti-MEM I Reduced serum medium | ||
| Line 14: | Line 14: | ||
* Cells | * Cells | ||
==Procedure - 6-well Format== | |||
'''1 day before transfection:'''<br> | '''1 day before transfection:'''<br> | ||
# 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 | # 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. | ||
'''Transfection:'''<br> | '''Transfection:'''<br> | ||
# | # At your bench, bring '''2.0 μg total plasmid DNA''' up to 20 μL total volume with fresh, sterile DNase-free water in fresh, sterile 1.5 mL tubes. Take the DNA samples to the tissue culture room. | ||
# In the tissue culture room, warm antibiotic-free growth medium (e.g., 10% FBS in McCoy’s 5A for, no pen/strep) at '''37°C'''. | |||
# Warm the Lipofectamine LTX vial, PLUS reagent vial, and and a 6 mL aliquot of Opti-MEM I Reduced Serum Medium to '''room temperature''' in the biosafety cabinet. | |||
# Prepare DNA-Lipofectamine complexes (600 μl per sample) as follows: | # Prepare DNA-Lipofectamine complexes (600 μl per sample) as follows: | ||
## Label sterile microfuge (1.5 ml) tubes. | ## Label sterile microfuge (1.5 ml) tubes. | ||
## 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 '''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 '''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. | # Add the total 600 μL of DNA+Opti-MEM+PLUS reagent+Lipofectamine mixture '''drop-wise''' to each appropriate well of cells. | ||
# Incubate cells at 37°C in a CO<sub>2</sub> incubator | # 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. | # (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 | Transgene expression should be detectable after 18 hours. | ||
<br><br> | <br><br> | ||
''For stable cells lines:'' Passage cells at a 1:10 (or higher) dilution into fresh growth medium | ''For stable cells lines:'' Passage cells at a 1:10 (or higher, e.g. 1:20, 1:40, etc.) dilution into fresh antibiotic-free growth medium 48 hours after transfection. The next day (after 18 -24 hours later), add selective medium (based on the resistance marker for your plasmid). Grow the cells for 1 - 2 weeks until isolated colonies (about 100 cells per colony) appear. Transfer single colonies into 24-well plates. Grow the cells until they reach 100% confluency. Expand the cells in larger growth vessels (e.g., 6-well plates or 10 cm plates) for further testing. | ||
Revision as of 09:45, 6 June 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
Procedure - 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.
Transfection:
- At your bench, bring 2.0 μg total plasmid DNA up to 20 μL total volume with fresh, sterile DNase-free water in fresh, sterile 1.5 mL tubes. Take the DNA samples to the tissue culture room.
- In the tissue culture room, warm antibiotic-free growth medium (e.g., 10% FBS in McCoy’s 5A for, no pen/strep) at 37°C.
- Warm the Lipofectamine LTX vial, PLUS reagent vial, and and a 6 mL aliquot of 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.
- 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 the total 600 μL of DNA+Opti-MEM+PLUS reagent+Lipofectamine mixture drop-wise to each appropriate 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 18 hours.
For stable cells lines: Passage cells at a 1:10 (or higher, e.g. 1:20, 1:40, etc.) dilution into fresh antibiotic-free growth medium 48 hours after transfection. The next day (after 18 -24 hours later), add selective medium (based on the resistance marker for your plasmid). Grow the cells for 1 - 2 weeks until isolated colonies (about 100 cells per colony) appear. Transfer single colonies into 24-well plates. Grow the cells until they reach 100% confluency. Expand the cells in larger growth vessels (e.g., 6-well plates or 10 cm plates) for further testing.
