3 replies to this topic

[flower]

Hi,

I've been trying to co-transfect siRNA and DNA into HeLa cells but so far have had no luck. I've tested DharmaFECT Duo and jetPRIME but had no success. I've had a little (but not good enough) success with sequential transfections, i.e. siRNA transfection using DharmaFECT1, followed 24 hours later by DNA transfection using FuGene. I've tried altering DNA and siRNA concentrations but to no avail.

If anyone has any advice/tips I'd be most grateful!

[Curtis]

my friends with Dharma have also failed.why not try using Fugene itself from the beginning? it worked fine for me. it gave me 99% transfection in HeLa for my pEGFP.N2 controls.

what is your DNA? you sure it's the circular plasmid? not contaminated with linear and nicked?

Edited by Curtis, 15 February 2011 - 06:43 AM.

[scolix]

try Lipofectamine. We use it routinely and get good knockdown.

[flower]

Curtis, on 15 February 2011 - 06:40 AM, said:

my friends with Dharma have also failed.why not try using Fugene itself from the beginning? it worked fine for me. it gave me 99% transfection in HeLa for my pEGFP.N2 controls.

what is your DNA? you sure it's the circular plasmid? not contaminated with linear and nicked?

I've never heard of anyone using FuGene for siRNA but I'm willing to give it a go. The DNA is good quality circular plasmid which I can get to express fine on its own, just not with the knock-down (it's an siRNA resistant plasmid so shouldn't be affected by the siRNA). Sometimes I get expression but no knock-down and sometimes get knock-down but little/no expression. I've tried knock-down prior to DNA transfection, DNA transfection prior to siRNA, both together, varying amounts... Maybe it is a transfection reagent problem.