I need to quantify the amount of relic/free DNA in water samples from a fish pond. I'm looking for a protocol with these method. Does anyone know of any accurate methodology for this quantification?
Cheers,
Caio
You can extract the total DNA from water using any commercially available kit. The extracted DNA can be quantified using nanodrop spectrophotometer. And thus the total DNA per unit volume of water can be determined.
If you want to qualify some specific DNA strand in the sample you can go for quantitative real time PCR using the extracted DNA.
Thanks Sutanu Karmatar!
I need to know the amount of relic/free DNA in water samples before making the DNA extraction. I thought of using the nanodrop to measure the amount of DNA sample before and after extraction. But the nanodrop doesn't provide a very accurate result.
Hi Caio Cesar Pires de Paula ,
There are more robust commercially available kits that can be used to extract DNA and are much more accurate, albeit costly, than the traditional nanodrop approach. We have used these kits in the past to sample microbial mats from extreme environments where sample is precious and requires special handling: https://www.thermofisher.com/order/catalog/product/Q33120?SID=srch-srp-Q33120 .
We ended up needing a very robust measurement because similar to Sutanu Karmakar recommendation, we needed to know the starting concentration of DNA prior to running quantitative analysis. Rather than use qPCR, which provides a relative measurement and requires a standard curve with your target genes (and is tough with 16S rRNA where there are multiple copies) we used droplet digital PCR which provides absolute quantification. This is a relatively new technology patented by Bio-Rad but it's worth looking into if you're going to be doing this type of analysis regularly. May I also recommend using the MoBio PowerWater kit for your extractions, I used a similar one in a similar ecosystem and it worked quite well.
All of this being said, if your plan is to sequence the DNA eventually, many sequencing facilities often require a nanodrop reading as part of their standard protocols. There is no reason you could not also quickly provide this as well.
Good luck and I'd be happy to try and answer any other questions in this thread!
Daniel
- Badges
- Science method
- Similar topics
- Methodology