Has anyone got any experience of transfecting plasmids into MG-63 using Lipofectamine 2000? I am currently using it to transfect three GFP-containing plasmids - one of which is 7kb and the other two are 9kb. At the moment, the protocol I use for a 24 well plate per well is: 2µl L2000 + 1µg DNA, increasing the incubation of the complex for 30 mins rt before adding to the medium (w/out antibiotics). I find that the 7kb plasmid transfects a lot better than the 9kb, where I only get a few cells.
Any help would be much appreciated.
With MG-63 we've also noticed that larger plasmids can have trouble during transfections - a complex condenser tends to help (see https://altogen.com/product/mg-63-transfection-reagent-osteosarcoma-crl-1427/). There are other potential issues that relate to your transfection controls; do you have a control vector to assay for overall expression? GFP transfection for example would help you normalize transfection efficiencies to see if there are any substantial discrepancies among individual runs.
Ted Bremmer yes I have a positive transfection control with GFP and my empty plasmid (containing GFP) transfects quite nicely. It’s when I use the plasmids with GFP + my protein of interest where I get issues. The empty plasmid is 7kbp and with my protein of interest it’s 9.2kbp.
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