transfection - (Oct/20/2016 )
I'm trying to transfect a GFP tagged plamid bought from Origene to establish a stable cell line. The total size of plasmid from Origene is 9.8kb. Just for reference, the size of ORF of gene of interest is 3231bp.
I transfected HCT116 cells with
1. the target GFP-tagged plasmid from origene
2. another GFP-tagged plasmid as a positive control.
The cells were 80% transfected with the positive control plasmid (also GFP tagged) as observed by green fluorescence under a microscope.
However, the cells transfected with GFP-tagged plasmid from Origene showed no green fluorescence under the microscope.
Could the plasmid from Origene be too big to transfect? Should I subclone it into pcDNA3.1 vector?
Any advice would be appreciated.
Just for ref, the positive control plasmid which is also GFP tagged is 7.6kb.
No, this plasmid isn't too big to transfect. It may be that you need to play with the transfection conditions (amount of DNA, ratio of DNA:transfection reagent) or that you need to induce the expression somehow. What promoter is the Origene plasmid running and have you checked to see that the gene sequence is intact? Subcloning may work or it may not, but is probably worth attempting.