Customer Transfection Protocol
Cell Line: L02 (immortal hepatic cell line)
Transfection Reagent: X-tremeGENE HP reagent
Type of transfection: transient
A. Plate cells
Plate L02 cells in a volume of 2,000µl medium per well in a 6-well gelatin coated plate 20-24 hours before
transfection. Cells were 70-80% confluent prior to transfection.
B. Prepare X-tremeGENE reagent : nucleic acid complex
For normal transtection
1. Warm X-tremeGENE HP DNA Transfection Reagent to +15 to +25°C and vortex gently before use.
2. Place 100 µl of Opti-MEM I Reduced-Serum Medium in a sterile tube.
3. Add 2 µg of DNA (3:1 ratio of reagent to DNA)
4. Pipet gently (10-15 times) to mix completely.
5. Add 6 µl of X-tremeGENE HP DNA Transfection Reagent to the diluted DNA.
6. Pipet gently (10-15 times) to mix completely.
7. Incubate at +15 to +25°C for 10-15 minutes for complex formation.
For low expression plasmid
1. Warm X-tremeGENE HP DNA Transfection Reagent to +15 to +25°C and vortex gently before use.
2. Place 100 µl of Opti-MEM I Reduced-Serum Medium in a sterile tube.
3. Add 5 µg of DNA (3:1 ratio of reagent to DNA)
4. Pipet gently (10-15 times) to mix completely or by vortex for a few seconds.
5. Add 15 µl of X-tremeGENE HP DNA Transfection Reagent to the diluted DNA.
6. Pipet gently (10-15 times) to mix completely or by vortex for a few seconds.
7. Incubate at +15 to +25°C for 10-15 minutes for complex formation.
C. Distribute the complexes to cells
1. Add 100µl of the X-tremeGENE HP Reagent : DNA complexes (prepared in Step B) drop-wise to
different areas of one well.
2. Gently rock the culture vessel back-and-forth and side-to-side to evenly distribute the X-tremeGENE HP
Reagent : DNA complexes.
3. Incubate for 20-24 hours.
D. Transfection results
Result 24 hours after transfection for L02 cells
Note:
RNAi plasmid contains GFP as a reporter .