2 replies to this topic

[anonymous]

i am  trying to  express  a  cloned  gene in  mammalian cells.i  have  tried  3   different  cell  lines  and  two different methods also.one  is  calcium  chloride and  the  other  is  QIAGEN trasfection reagent EFFECTENE.after trasfection i  am  doing  a  western to confirm  the presence of  protien but i  am  not  getting any  signal..toghether with this i am using a GFP vector  as  a  reporter.there also i  can  see  only  very  few  cells expressing  GFP.can  anybody out there give  me  any suggestions?

[anonymous]

First: Why is your promoter and Are in your cell type the necessary factor for your expression? For example: SV40 will need T Antigen.Second: Are you doing stable or transient transfections?

[anonymous]

Effectene should be quite good for a wide range of cells. The problem may be your plasmid constrution, or inappropiate GFP control. Try clontech's pEGFP-N1.