October 23, 2013
Transfection of KAH126
| Plate-Well |
Plasmid |
DNA |
Volume |
dH2O |
Lipo |
PLUS reagent |
Opti-MEM (total)
|
| 1-2 |
KAH126 |
1.5 μg |
23 μL > 20 μL |
0μL |
2.5μL |
7.5 μL |
570 μL
|
- 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.
Cell Culture of KAH126
Grow liquid culture of KAH126
- Label 15 ml sterile culture tubes. Fill each tube with 2 ml LB growth medium with Amp.
- Use sterile pipette tip to touch bacterial streak and put the tip into LB medium.
- Grow the cultures for 10-12 hrs.
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