I have been trying different protocols for genomic DNA extraction from bovine semen samples. Given the fact that in the lab we can not use solvents for DNA extraction, at the beginning I tried the Quiagen Blood&Tissue kit with some modifications. This protocol did not work ok, I had a lot of protein contamination, even if I have treated my samples with Dtt and proteinase K.Moreover I tried different periods of incubation (3h-overnight) and different proteinase K concentrations, but the protein contamination is still there (ratio 260/280 less or equal 1.5 and ratio 260/230 inferior to 1).
Reading some articles in forensic science I found the chelex 100 as a easy method for DNA extraction, so I did some extractions, and apparently it was working the ratio in nanodrop are ok, but sometimes I found the ratio 260/280 really high (5-7), so i started thinking that somethig was wrong with this product. I called the Bio-Rad technical support, and they told me that DNA can not be quantify by nanodrop because the chelex 100 does not eliminate the proteins and RNA
. Finally I decide to go to other lab and try the trizol and phenol-chloroform protocols. With the Trizol I have a lot of difficulties to dissolve the DNA pellet, and with the phenol chloroform I still have protein contamination even if I am using DTT and proteinase k before the extraction. I think that I have tried the most of available protocols for DNA semen extraction, and they are not working at all. I need a pure DNA because i need to quantify the presence of a multi-copied gene. I dont know what to do, I am desperate
. Do you have some suggestions?