Haynes:TransfectionPlasmid Lipo

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<- [[Haynes:Protocols | Back to Protocols]]
<- [[Haynes:Protocols | Back to Protocols]]
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=Mammalian Cell Transfection - Lipofectamine LTX Reagent=
=Mammalian Cell Transfection - Lipofectamine LTX Reagent=
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'''1 day before transfection:'''<br>
'''1 day before transfection:'''<br>
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# 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.
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# 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>
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# Warm plasmid DNA, Lipofectamine LTX, PLUS reagent, and Opti-MEM I Reduced Serum Medium to room temperature in the biosafety cabinet.
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# 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.
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# 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'''.
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# 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.
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## 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 '''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 '''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.
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# Add DNA+Opti-MEM+PLUS reagent+Lipofectamine mixture '''drop-wise''' to each well of cells.
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# 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.  
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Transgene expression should be detectable after 24 hours.
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Transgene expression should be detectable after 18 hours.
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<br><br>
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''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.
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''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.

Current revision

<- Back to Protocols

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:

  1. Seed cultures in 6-well plates at ~2.5x105 so that cells will be ~5.0x105 at the time of transfection.

Transfection:

  1. 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.
  2. 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.
  3. 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.
  4. Prepare DNA-Lipofectamine complexes (600 μl per sample) as follows:
    1. Label sterile microfuge (1.5 ml) tubes.
    2. Add 570 μL Opti-MEM to each 20 μL DNA sample.
    3. Add 2.5 μL PLUS reagent to each DNA+Opti-MEM sample. Incubate for 5 minutes at room temperature.
    4. Add 7.5 μL Lipofectamine LTX to each DNA+Opti-MEM+PLUS reagent sample. Incubate for 30 minutes at room temperature.
  5. Add the total 600 μL of DNA+Opti-MEM+PLUS reagent+Lipofectamine mixture drop-wise to each appropriate well of cells.
  6. Incubate cells at 37°C in a CO2 incubator
  7. (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.
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