Haynes:TransfectionPlasmid Lipo

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(New page: <- Back to Protocols <div style="width: 800px" align="center"> '''Mammalian Cell Transfection - Lipofectamine Reagent'''<br> Karmella Haynes, 2012 </div> <div styl...)
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# Warm plasmid DNA, Lipofectamine 2000, and Opti-MEM I Reduced Serum Medium to room temp.  
# Warm plasmid DNA, Lipofectamine 2000, and Opti-MEM I Reduced Serum Medium to room temp.  
# Prepare DNA-Lipofectamine complexes (500 μl per well) as follows:
# Prepare DNA-Lipofectamine complexes (500 μl per well) as follows:
-
## Label sterile microfuge ml tubes.
+
## Label sterile microfuge (1.5 ml) tubes.
-
## Dilute plasmid DNA (4 μg) in 250 μl of Opti-MEM in each tube.
+
## Dilute '''plasmid DNA (4 μg)''' in 250 μl of Opti-MEM in each tube.
## Dilute 4.0 μl Lipofectamine 2000 in 250 μl of Opti-MEM (per sample). Incubate for 5 minutes at room temp. Do not let the mixture sit for more than 25 minutes.
## Dilute 4.0 μl Lipofectamine 2000 in 250 μl of Opti-MEM (per sample). Incubate for 5 minutes at room temp. Do not let the mixture sit for more than 25 minutes.
## Add 50 μl of the diluted Lipofectamine 2000 to the diluted DNA (total volume = 100 μl). Mix gently and incubate for 20 minutes at room temp. Complexes are stable for 6 hours at room temp.
## Add 50 μl of the diluted Lipofectamine 2000 to the diluted DNA (total volume = 100 μl). Mix gently and incubate for 20 minutes at room temp. Complexes are stable for 6 hours at room temp.

Revision as of 22:42, 16 November 2012

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Mammalian Cell Transfection - Lipofectamine Reagent
Karmella Haynes, 2012

Note: Good for quick-and-dirty transfections; pretty toxic

Materials

  • Lipofectamine 2000
  • Opti-MEM I Reduced serum medium
  • Antibiotic-free growth medium (no pen/strep or other antibiotics)

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. Use antibiotic-free growth medium (e.g., 10% FBS in McCoy’s 5A for, no pen/strep).

Transfection:

  1. Warm plasmid DNA, Lipofectamine 2000, and Opti-MEM I Reduced Serum Medium to room temp.
  2. Prepare DNA-Lipofectamine complexes (500 μl per well) as follows:
    1. Label sterile microfuge (1.5 ml) tubes.
    2. Dilute plasmid DNA (4 μg) in 250 μl of Opti-MEM in each tube.
    3. Dilute 4.0 μl Lipofectamine 2000 in 250 μl of Opti-MEM (per sample). Incubate for 5 minutes at room temp. Do not let the mixture sit for more than 25 minutes.
    4. Add 50 μl of the diluted Lipofectamine 2000 to the diluted DNA (total volume = 100 μl). Mix gently and incubate for 20 minutes at room temp. Complexes are stable for 6 hours at room temp.
    5. Add 500 μl of complexes to each well. Mix with gentle rocking.
  3. Incubate cells at 37°C in a CO2 incubator for 5-6 hours. Wash cells once with warm 1xPBS. 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.
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