Hi,

I'm trying to find a pDNA transient transfection carrier for my MDA-MB-231.

I'm using either liposome or lipopolyplex, but the transfection variability between experiments are too great.

I can only suspect that thin film hydration method has high variability (due to water bath sonication) and changed to ultrasonication which gave 0% transfection (positive ctrl worked, so no probs with pDNA).

Here's the protocol.

1) Thin film made in 4-mL vial or RB or e-tube using rotovap (5 mg/mL lipids in chloroform)

2) Hydration tested with DW/opti-mem/PBS/HEPES using water (1 mg/mL)

3) DNA solution added to liposome solution while vortexing

4) Cells treated with 1 ug DNA/well in opti-mem for 4 hrs, then complete media exchanged or added

For lipoplexes,

1) LPEI solution was added to DNA solution (N/P=10), RT incubation, 30 min

2) Liposome mixture thin film made as above

3) Hydration tested with various buffers or polyplex solution

4) Polyplex solution added to liposome

5) Cells treated with 1 ug DNA/well in opti-mem for 4 hrs, then complete media exchanged or added

I'm already on a number of tries and been frustrated with the result because no matter how consistent I am, the results are different.

Please share your wisdom with me!