Help! low efficiency of transfection - (Sep/15/2010 )

Hi everyone, I'm a new Ph.D.student and i'm stuck with transfection step for many months. Could you please help?

I try to transfect PreB cells which are suspension cells using fugene6 and my DNA is EGFP but i got very low efficiency.

I had been varied cell density, fugene:DNA ratio but not thing better.

I usally use Optimem for dilute fugene reagent and incubate 20-35 mins.

My supervisor told me that even summer student can do this transfection at least 50% efficiency.

Should i try to vary an incubation time to up to 2hrs (from a company trouble shooting guide)?

I had email to a couple of company, most of them told me to use electroporation instead but my supervisor had tried with these cell lines before and she told me that more than 50% of our cells will dead.

The warning on the little bottle with fugene says that you should not get it into contact with the plastic walls of the tubes you pipet it in; you need to pipet it directly into the medium/dna solution. Hope this helps. If you have no luck with this, an alternative method of transfection is with PEI (http://www.ncbi.nlm.nih.gov/pubmed/10467362).

Check your cells for Mycoplasma contamination. If so You wont get much efficiency