2 replies to this topic

[sgylee]

Hello, I need some advice because I am stuck....

I am trying to knockdown c-Myc and/or RAS using miRNA, let-7.

This is basic protocol I used.

- 12-well plate
- 150,000 cells per well. (HeLa, A549, Panc-1, MB231)
- Total transfection volume: 1250 microL (1000 microL of growth and 250 microL of diluted medium)
- 80nM of miRNA per well
- Tried 1, 2, and 4 microL of Lipofectamine 2000
- 2 days transfection

Need advice on what I should try next.

Thank you very much

[Fizban]

sgylee, on Feb 6 2010, 06:43 PM, said:

Hello, I need some advice because I am stuck....

I am trying to knockdown c-Myc and/or RAS using miRNA, let-7.

This is basic protocol I used.

- 12-well plate
- 150,000 cells per well. (HeLa, A549, Panc-1, MB231)
- Total transfection volume: 1250 microL (1000 microL of growth and 250 microL of diluted medium)
- 80nM of miRNA per well
- Tried 1, 2, and 4 microL of Lipofectamine 2000
- 2 days transfection

Need advice on what I should try next.

Thank you very much

can't help without details....what is the problem here?
why are you stuck?
what do you mean when u say 80nM mirna? single strand synthetic? overexpressing vector? commercial mimics?
try to help us helping u!  
Fiz

[j123]

Try NeoFX transfection reagents from AB.  It works very well for me.  It also depends on different cells.  Good luck!

sgylee, on Feb 6 2010, 12:43 PM, said:

Hello, I need some advice because I am stuck....

I am trying to knockdown c-Myc and/or RAS using miRNA, let-7.

This is basic protocol I used.

- 12-well plate
- 150,000 cells per well. (HeLa, A549, Panc-1, MB231)
- Total transfection volume: 1250 microL (1000 microL of growth and 250 microL of diluted medium)
- 80nM of miRNA per well
- Tried 1, 2, and 4 microL of Lipofectamine 2000
- 2 days transfection

Need advice on what I should try next.

Thank you very much