DNA-PEI in anionic liposomes, picogreen quantification

[limor]

Hi everyone
In my research I produce plasmidial DNA-PEI complexes, which later I entrap into anionic liposomes.
In order to quantify the entrapped DNA, I ultra-centrifuge the liposomes, then re-suspend them in 0.1% Triton X100 for 30 minutes to break down the cells and then add Heparin to 0.3% for another 30 minutes in order to release the DNA from the PEI.
I then quantify the DNA using PicoGreen. In addition, I quantify the DNA that was not entrapped, in the supernatant from the ultracentrifuge.
The problem is, the DNA in the liposomes plus the DNA in the supernatant don't add up to the amount of the DNA I added in the first place!
Is there a problem in my method/concentrations/ times?

Thank you all!
Limor

[CrowSan]

Hi - in short I'm not sure what is wrong. I assume you are measuring the DNA based on fluoresence (plate reader)?
The excess "DNA" may be from background fluoresence from unbound dye.
This paper here (which I have not read) mentions that pico green and Heparin give a fluorescent background:
http://www.babonline.org/bab/036/0013/0360013.pdf
If you are using a plate reader you may need to "blank" it against a well containing the heparin/pico green solution that you use to release the DNA (without DNA present).
Sorry I can't be more help
CrSn

[limor]

Thank you very much for your help! i will read it.
Limor