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Plasmid DNA - (Jun/24/2013 )

Dear All
i am hoping that you are fine.
this is my first post
i have several questions about working with plasmid DNA
so please help me if you can
1- N/P ratio
i am working on formation of polyion complex between cationic polymer and pDNA. I want to calculate exact N+ and P- exactly. can you tell me how i do it? because i dont have any idea about it.
2- gene expression
I am measuring gene expression for prepared complex but i donot get higher expression even with using PEI polymer as control.
one of reasons:
a- degration of pDNA but result of gel electrophoresis is only one band not several bands.
can you tell what i should to do else to confirm the condition of my plasmid?
waiting for your urgent reply


Dear All
does any one has a clue for what I am saying?


No, we don't. I'd vaguely guess you are looking at using cationic compounds as a way of improving transfection efficiency, but the details of what you are asking remain completely opaque.


Dear Phage434
yes you are right
N/P ratio
N: mole of amine group of polymer
P: mole of phosphate group of DNA
I prepared complex between polymer and DNA according to different N/P ratios
so I want to know the exact amount of amine group of polymer that attached to phosphate group of DNA you can say N+/P- (exact amount used).
Also, I want to know how I can check the condition of plasmid, since it doesnot produce high light units by using luciferase assay kit, which didnot confirm its expression.
I performed gel electrophoresis for plasmid and I got one band but if i got several bands I guess that pDNA is degraded but when I measure its concentration it is 850ng/ul.
please help me