6 replies to this topic

[lyok]

Hallo all,

I am wondering if anyone here knows a good method to label or stain DNA that I would like to inject in cells.
I want to verify if the injected DNA stays inside the cell. I wanted to do this by labeling the DNA (its just random plasmid DNA) but I dont know what would be a good kit to use.

Any ideas?

[bob1]

How would you like to detect it?  You could radio-label, fluorescent tag, DIG tag etc.

[lyok]

bob1, on 10 February 2013 - 02:33 PM, said:

How would you like to detect it?  You could radio-label, fluorescent tag, DIG tag etc.

I think I would prefer a fluorescent tag.
DIG tag is not something I can use I think because then I have to add something else that can react with the DIG? (I am not that familiar with DIG labeling).
I really need DNA that is labeled that I can inject and then check whether its in the cell or not.

[bob1]

Actually I think the easiest way to do it would be to use a vector that expresses GFP (perhaps independently of the gene of interest) and look for expression in the live cells.  I think all other methods would require you to kill the cells to detect the plasmid or some output of the plasmid.

There are a number of companies that sell fluorescently labeled plasmids for this sort of purpose.

DIG works by using a highly specific anti-DIG Fc fragment, similar to doing  normal immunofluorescence.

[lyok]

bob1, on 10 February 2013 - 11:42 PM, said:

Actually I think the easiest way to do it would be to use a vector that expresses GFP (perhaps independently of the gene of interest) and look for expression in the live cells.  I think all other methods would require you to kill the cells to detect the plasmid or some output of the plasmid.

There are a number of companies that sell fluorescently labeled plasmids for this sort of purpose.

DIG works by using a highly specific anti-DIG Fc fragment, similar to doing  normal immunofluorescence.

Yes, but for that to work, I need a promoter that is active in the cell, and there arent any as far as I know.
Thats why I was wondering if there were any good labeled DNA kits.

[bob1]

I'm pretty sure Invitrogen/Life Technologies do sell this sort of DNA (they certainly sell fluorescent controls for many siRNA sorts of things), but whether they sell a kit with the capacity to label your plasmid I don't know.  I'd ring your local rep and/or tech support and see.

[lyok]

bob1, on 11 February 2013 - 12:41 AM, said:

I'm pretty sure Invitrogen/Life Technologies do sell this sort of DNA (they certainly sell fluorescent controls for many siRNA sorts of things), but whether they sell a kit with the capacity to label your plasmid I don't know.  I'd ring your local rep and/or tech support and see.

Oh ok.
I hoped you could buy it premade.