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[cnoakes84]

Hi there

I'm having real difficulty in generating a stable MCF10A cell line that expresses the TetR in Clontech's Tet-ON system.

Currently, I am electroporating ~1.5 x10⁶ cells with 10 ug of linear (ScaI cut) or circular TetON-Advanced vector, replating them in 10 cm dishes, allowing them to grow for 48 hours before selecting with 200 ug/ml G418 (calculated from cell death curve).

I get approx 10-20 colonies growing in selection after 2 weeks. My negative control is mock electroporated cells with no DNA and I get no colonies growing in selection after 2 weeks. I then pick the colonies and assay them for TetR expression by transiently transfecting a tet-responsive plasmid which should express GFP when induced with dox. I also co-transfect MCherry plasmid to ensure I am transfecting the cells with the TRE plasmid.

I have screened over 100 colonies and none have GFP expression when tested. I know the Tet-ON plasmid is ok, as in transient transfections and inductions I see GFP expression.

I have also managed to get a clone which has the tet-resposive element (expressing GFP) stably integrated into the MCF10A genome and is HygroB selective. When I transiently transfect in the Tet-ON plasmid and induce in these cells then I see GFP expression. This cell line was generated by transfecting the cells with 10ug of the plasmid (linearised) with Mirus' TransIT LT reagent.

I have also tried to get the TetON plasmid in this cell line, but when select with G418 + Hygro B I dont get any colonies growing!

If anyone has any suggestions/experience in generating Tet-ON driver lines in any cell line, or more specifically in MCF10A cells then please let me know! I want to preserve what hair I have left.......

[kay]

Hello, Please I am new to this forum. pardon me if i posted this on a question already being asked.

I am having problems with my pcr. the methodology i am using says add 0.64micromol of each of the dntps. But my dntp reagent is labelled 'dntp mix'. Am i supposed to add four times the volume (v1) i get from my c1v1=c2v2 equation? Or is it that v1 contains equal concentrations of each of the nucleotides? Someone help!