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DNA extraction from semen samples

DNA extraction from semen samples - DNA Techniques

DNA extraction from semen samples - Post questions and discuss DNA techniques and protocols such as DNA extraction, PCR, and the study of DNA-binding proteins.


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  #1  
Old 11-02-2011, 09:13 PM
Pipette Filler
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Red face DNA extraction from semen samples



Hello everybody,
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?
Thanks
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Old 11-16-2011, 04:08 AM
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Default Re: DNA extraction from semen samples

Try sonicating the cells before using the Trizol protocol.
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Old 12-02-2011, 04:11 AM
Pipette Filler
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Default Re: DNA extraction from semen samples

Quite right!Maybe it will be successful!Add one scentence:You are excellent!
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Old 12-05-2011, 08:48 PM
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Default Re: DNA extraction from semen samples

Thanks,
I am going to try the sonication technique good idead. Until now, the best protocol that I found it is phenol chloroform, but I would like to find a protocol without the using of solvents.
Alyssaly I try to repeat myself all the time that I am excellent but when the things are not working is not so evident :P.
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