2 replies to this topic

[Chuvy]

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.

[hematopoietry]

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....pubmed/10467362).

[ranvi]

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