DNA extraction from bovine semen
I am doing a postdoc, and during the last months I have been trying different protocols for DNA extraction in semen samples. Given the fact taht in the company I can not use solvents (Trizol, Phenol-chloroform) for DNA extraction, I tried several protocols without using solvents, I used two user developed protocols from Qiagen, consists in using the the blood tissue kit with two different lysis buffers (home made) with DTT, proteinase K and guanidinium. The protocol works only for few bulls, and I assume that it is only with the old bulls, which maybe have a weak protamines. The problem is that with the young bulls I can not obtain DNA from sperm with good parameters, I obtaine ratio under the optimal less than 1.5 for 260/280 ratio, and les than .5 for 260/230 ratio.Moreover the concentration of DNA is very low, so I think that the aforementioned it s the result of a protein contamination.
I found some forensic articles, which talk about the use of chelex 100 to purify the DNA in semen samples, so I tried this technology, and I was able to obtain DNA but the paramters measuring DNA in the nanodrop were really weird (260/280 around 5-8), so I call the company, and they explain me that the chelex works only to purify the DNA, but you can not quantify the DNA, not at least by nanodrop.
Finally I decided to go to another lab, (this lab allows the use of solvents) and I tried trizol and phenol-chloroform protocols. With trizol I obtained a good pellet at the end of the process but I was unable to dissolve the pellet in TE and even in a mild base (8mM NaOH) as the protocol suggest. With the Phenol-Choloform I had better results i have more DNA concentration a higher 260/230 ratio (1.2-1.8) but the ratio 260/280 is still low around 1.62.
I was wondering if somebody has the same problem or if you have other protocols. I feel very frustrated because I have been working really hard in this project i dont obtain the expected results.:mad:
|All times are GMT. The time now is 01:15 AM.|
Powered by vBulletin® Version 3.8.4
Copyright ©2000 - 2015, Jelsoft Enterprises Ltd.
Copyright 2005 - 2012 Molecular Station | All Rights Reserved