Stable transfection - (May/10/2012 )
1. How long would this process take (matter of weeks/months/few months?)
2. What is the success rate of the process?
3. If my vector does not have a GFP tag, how would I select out the positively transfected cells?
4. Would larger plasmid = tougher transfections (FYI, I have a 16kb plasmid)
1) depends on the selection process, typically the longest part (if making monoclonal lines - not the anitbody type) is the growing up from single colonies. This may take a month or two. Polyclonal lines can be made in a couple of weeks.
2)Pretty high if things are optimised before hand. Making monoclonal lines I was able to grow up about 70% of the colonies to a point where they were viable as lines, and had about 95% test positive for the insert.
3)The positive cells should survive selection... you can then test for expression f the gene by PCR or western/IF.
4)Often yes, I think this is due to the relatively low copy number of the plasmid in the same concentration of DNA, and the increased chance of degradation due to the cell digesting the foreign DNA.
Does your vector contain any sort of integrase elements that help integration or is selection with an antibiotic your method?