I'm working on developing a method to extract and isolate DNA from respiratory condensate or "blow" samples collected from beluga whales. In the field, we tried 2 different collection devices, a swab and nitex membrane. The nitex membrane was secured over a petri dish using a rubber band and the sample was collected on to the membrane. It was then placed inside of a 50 ml tube with desiccant to dry. The membrane is about 15 cm across, making it too large to use the suggested amount of ATL and Proteinase K for extractions. My question is, what's a good way to remove the dried cells/DNA from the nitex membrane so that I can continue to use a DNeasy kit? Any suggestions would be greatly appreciated!!
Hi Justine,
Can you see where on the membrane the dried cells are? If so I would cut the membrane to only those parts as a first step. Indeed the protocol volumes of ATL/ProK won't be enough. However, what about keeping the protocol ratio for those two substances, but increasing the total volume so that you can (partially) submerge your membrane, and then run the first column step several times. Since its the same sample you are not contamnating the column' filter, and the DNA should attach to the filter I believe, even when you would do it several times.
Be carefully that you don't put too much DNA on the filter, since it will then get clogged. You probably don't even need to collect all dried cells to get DNA.
OR
You cut the membrane in very small pieces and incubate them in the same ATL/ProK solution per batch, so you retain the protocol volume and accumulate DNA from multiple smaller membrane parts.
Just some first thoughts. Good luck
Matthijs P. van den Burg Ali Mahmoudpour Thank you! I think I'll try a combination of both of your answers. I might use phenol chloroform to extract but I need to find out how it reacts with nitex first.
As mentioned by Ali Mahmoudpour you can get better yields of DNA by immersing the membrane in milli Q water and then go for phenol chloroform instead of TE.
- Badges
- Science topic
- Similar topics
- Phytochemistry
- Extracts