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DNA-PEI in anionic liposomes, picogreen quantification


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#1 Limor Sertshuk

Limor Sertshuk

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Posted 14 August 2012 - 10:01 AM

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

#2 bob1

bob1

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Posted 14 August 2012 - 12:34 PM

How big a difference are we talking here?

It is easily possible that you are losing some DNA by degradation by secreted DNases, or during the wash etc. steps. It is also possible that the sensitivity of your assays is not high enough, or is non-linear.




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