Using Picogreen to quantitate DNA - Questions about how to make this work! (Nov/26/2006 )

Hi All!

We have some environmental DNA samples that we need to quantitate. The problem is, they are very small, very dilute samples --- On the order of 0.1 ng/microliter (0.1 micrograms/ml) and below the threshold to accurately measure using OD260/280.

There is a reagent called Picogreen which binds double stranded DNA and fluoresces strongly. The manufacturer claims that it is 100 times more sensitive than traditional spectrophotometric methjods, to quantitate DNA. You simply add it to your DNA, put it on your transilluminator, and measure the fluoresence (similar to measuring band intensity in a gel.)

Here are my problems.

(1) First of all, you use this reagent in a 96-well plate. You need a plate with wells that are clear on the bottom and black on the sides (to prevent "crosstalk" between samples.) We've tried two different types of plates that meet this description, both from NUNC. One plate fluoresces on its own, so we stopped using that one. The second seems to have some background issues as well. My question here, is: Can you recommend a plate that would be approriate for this application (ie a plate that has a very low background fluorescence under UV light)?

The excitation of Picogreen is similar to Fluorescein and SYBR gold.

(2) A second thing we tried : We tried to use this reagent in the QPCR instrument as well. This got around the problem with the plates (the optical strips for QPCR are pretty good), but I am not certain how to set up the instrument to read Picogreen. What reporter would I choose, or what settings would I alter, to ask the instrument to use a different wavelength of light? Again, I want to shoot for the SYBR gold/Fluorescein settings, but I don't see those on the list of reporters.

Any help you can give, is appreciated. I'm also not sure how to set the cycles on the QPCR instrument, for this application. If you have ever tried anything like this, please let me know. And, if I'm not being clear, please let me know that as well.

I have used a similar procedure using the qPCR machine. I followed the following paper

Blotta, I., et al., Quantitiative assay for total dsDNA with picorgreen reagent and real-time detection.
Ann Ist Super Sanita 2005 41 (1) 119-123

http://www.iss.it/publ/anna/2005/1/411119.pdf

I had trouble however figuring out how to set up the machine I was using (ABI 7500). I then did a single plate read as can be set up on the ABI machine. This gave me raw flourescence data that I compared to a standard curve I ran at the same time. I had ok results with this, but not always 100% reliable for some reason I never figured out.