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Guinea-pig brain tissue (Trizol): Phenol contamination


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#1 claire101

claire101

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Posted 19 May 2011 - 11:41 PM

Hi all,

I am new to bioforum, so please dont mind me if I am asking questions that has already been answered multiple times. I am extracting RNA from guinea-pig brain and previous people before me had problems extracting guinea-pig brain RNA for some weird reason. Commerical kits were used before, but I am trying Trizol out as it has proven to give me high yields as well as good A260/A280 ratios. I have problems however with my A230/A260 ratios which indicates high phenol contamination. I understand from these forums, that because brain is high in lipid content, after homoegenising my tissue and doing the first spin prior to chloroform addition, I did notice the white lipid layer above my Trizol. My question is how do I get rid of it, as I read somewhere here too that any lipid carry over will carry phenol over too. My next question is I want to do a double chloroform extraction to get rid of phenol, how do you do this exactly? I know the first chloroform addition is 200ul for every 1ml of Trizol. Is the second extraction adding chloroform 1:1 directly to the aqueous phase after spinning down and then spinning again? My last question is whether ethanol precipitation really get rids of phenols? Basically I need to know how to work with brain tissue and tissue with high lipid as I keep getting phenol contamination. Thank you so much!




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