A happy Christmas and new year!
I am using TRIZOL to extract DNA from human cerebrospinal fuid (CSF). Generally, the number of cells in the CSF are very low (100-1000 cells/ml) and I am having trouble with the yield and quality of DNA. I use 800ul Trizol, 1 ul glycogen just before ethanol precipitation, and Na-acetate to wash the pallet. Then I use 75% ethanol to remove the salt and then resuspend in water. In the original protocol, resuspension should be with 8mM NaOH. Is this necessary or can I use water to resuspend?
Today, I made a mistake and instead of using 75% EtOH, I used 25% EtOH to wash the pellet. Since, the quantity of DNA is so low that I usually cannot see a pellet, and neither I can use a spectrophotometer to quantify the DNA, I am not sure if I have lost the DNA.
Besides this, does someone have a general advice to increase the yield of DNA from extremely low number of cells? These are human samples and I cannot play around.
P.S. In Na-acetate wash step, can I just do it once instead of two washes? I fear with each wash step I will loose DNA. Also, would 10% ethanol in Na-acetate solution not dissolve the DNA?
Edited by Yasar, 26 December 2011 - 07:51 PM.