I extracted leaf DNA and generally get good concentrations of DNA. My protein and carb ratios can be off a little at times, but I usually don't use if the nanodropper reports a negative value. I don't know if its my nanodropper, but I'm wonder if any have experience getting good PCR product with 260/230 values such as -1.26?
Steve,
If your spectrophotometer can print/display an absortpion curve from 220-340nm do it.
Plants have phenolic compounds that absorb strongly at short wave UV (remember organic chem?) It had no practical application in your life until now! Find a protocol that removes these phenolics.
You can't have a negative ratio with a functioning and correctly zero'ed spectrophotometer. Both absorbances must be zero or positive against your background buffer. Compounds either absorb light or they don't. A negative ratio would mean that at one but not the other wavelength, the compound absorbs less than zero, i.e. it emits light. That's too unlikely to be real to be even worth considering.
Most likely the problem is a bubble, insoluble contamination of the lens, or blanking against a buffer with strong background absorbance at those wave lengths. Try scrubbing it really hard with Kim wipes and dish detergent.
The cost of running that PCR seems so low that you should just do it, see what happens...
Thank you gentlemen. I appreciate your insight. I knew it couldn't actually be a negative ratio, and would repeat the recording after re-blanking and at times it would...anyway, we believe it is contamination of the TE buffer I'm using as three others in the lab have pulled from it as well. However, David Spencer, 0.1mM may be something I'll try - thanks.
They were older leaves and had more phenols...we thought of that, but hadn't had the problem before.
Unfortunately, John, my grad adviser is VERY frugal. I will add for what it is worth, that after letting the sample sit overnight (4 C) the values were fine... go figure.
Thanks for the input - will follow the tips tomorrow
I would rather says if it is not the issue with Nanodrop then off course it is the contamination of the sample.
- Badges
- Science method